scholarly journals Rapid Sepsityper in clinical routine: 2 years’ successful experience

2020 ◽  
Vol 69 (12) ◽  
pp. 1398-1404
Author(s):  
Miriam Cordovana ◽  
Anna Zignoli ◽  
Simone Ambretti
Keyword(s):  
Sample Preparation ◽  
Causative Agent ◽  
Type Species ◽  
Susceptibility Testing ◽  
Preparation Method ◽  
Bloodstream Infections ◽  
Routine Practice ◽  
Sample Preparation Method ◽  
Content Type ◽  
Link Type

Introduction. Rapid identification of the causative agent of sepsis is crucial for patient outcomes. Aim. The Sepsityper sample preparation method enables direct microbial identification of positive blood culture samples via matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS). Hypothesis/Gap statement. The implementation of the Sepsityper method in the routine practice could represent a fundamental tool to achieve a prompt identification of the causative agent of bloodstream infections, and therefore accelerate the adoption of the proper antibiotic treatment. Methodology. In this study, the novel rapid workflow of the MALDI Biotypr Sepsityper kit (Bruker Daltonik GmbH, Germany) was evaluated using routine samples from a 2-year period (n=6918), and dedicated optimized protocols for the microbial groups that were more difficult to identify were developed. Moreover, the use of the residual bacterial pellet to perform susceptibility testing using different methods (commercial broth microdilution, disc diffusion, gradient diffusion) was investigated. Results. The rapid Sepsityper protocol allowed the identification of 5470/6338 (86.3 %) monomicrobial samples at species level, with very good performance for all of the clinically most significant pathogens (2510/2592 enterobacteria, 631/669 Staphylococcus aureus and 223/246 enterococci were identified). Streptococcus pneumoniae , Bacteroides fragilis and yeasts were the most troublesome to identify, but the application of specific optimized protocols significantly improved their rate of identification (from 14.7–71.5 %, 47.8–89.7 % and 37.1–89.5 %, respectively). Specificity was 100 % (no identification was made for the false-positive samples). Further, the residual pellet proved to be suitable to investigate susceptibility to antimicrobials, enabling us to simplify the workflow and shorten the time to report. Conclusion. The Rapid Sepsityper workflow proved to be a reliable sample preparation method for identification and susceptibility testing directly from positive blood cultures, providing novel approaches for accelerated diagnostics of bloodstream infections.

scholarly journals Genomic investigation of a suspected Klebsiella pneumoniae outbreak in a neonatal care unit in sub-Saharan Africa

2021 ◽  
Vol 7 (11) ◽  
Author(s):  
Jennifer Cornick ◽  
Patrick Musicha ◽  
Chikondi Peno ◽  
Ezgi Seager ◽  
Pui-Ying Iroh Tam ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Type Species ◽  
Bloodstream Infections ◽  
Sub Saharan Africa ◽  
Content Type ◽  
Link Type ◽  
Sub Saharan ◽  
Neonatal Care Unit ◽  
Suspected Outbreak

A special-care neonatal unit from a large public hospital in Malawi was noted as having more frequent, difficult-to-treat infections, and a suspected outbreak of multi-drug-resistant Klebsiella pneumoniae was investigated using genomic characterisation. All K. pneumoniae bloodstream infections (BSIs) from patients in the neonatal ward (n=62), and a subset of K. pneumoniae BSI isolates (n=38) from other paediatric wards in the hospital, collected over a 4 year period were studied. After whole genome sequencing, the strain sequence types (STs), plasmid types, virulence and resistance genes were identified. One ST340 clone, part of clonal complex 258 (CC258) and an ST that drives hospital outbreaks worldwide, harbouring numerous resistance genes and plasmids, was implicated as the likely cause of the outbreak. This study contributes molecular information necessary for tracking and characterizing this important hospital pathogen in sub-Saharan Africa.

scholarly journals Molecular characterization of a novel bla CTX-M-3-carrying Tn6741 transposon in Morganella morganii isolated from swine

2020 ◽  
Vol 69 (8) ◽  
pp. 1089-1094
Author(s):  
Xingwei Luo ◽  
Yajun Zhai ◽  
Dandan He ◽  
Xiaodie Cui ◽  
Yingying Yang ◽  
...  
Keyword(s):  
Type Species ◽  
Susceptibility Testing ◽  
Morganella Morganii ◽  
Ionization Time ◽  
Content Type ◽  
Genetic Environment ◽  
Link Type ◽  
Flight Mass Spectrometry ◽  
First Time

Introduction. The bla CTX-M-3 gene has rarely been reported in Morganella morganii strains and its genetic environment has not yet been investigated. Aim. To identify the bla CTX-M-3 gene in M. morganii isolated from swine and characterize its genetic environment. Methodology. A M. morganii isolate (named MM1L5) from a deceased swine was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing. The bla genes were detected and then the genetic location and environment of bla CTX-M-3 were investigated by Southern blot and PCR mapping, respectively. The M. morganii bla CTX-M-3 gene was cloned and expressed in Escherichia coli . Results. Isolate MM1L5 harboured the bla CTX-M-3 and bla TEM-1 genes. The bla CTX-M-3 gene, located on the chromosome, was co-carried with an IS26 and bla TEM-1 gene by a novel 6361 bp IS26-flanked composite transposon, designated Tn6741. This transposon consisted of a novel bla CTX-M-3-containing module, IS26-ΔISEcp1-bla CTX-M-3-Δorf477-IS26 (named Tn6710), and a bla TEM-1-containing module, IS26-Δorf477-bla TEM-1-tnpR-IS26, differing from previous reports. Phylogenetic analysis showed a significant variation based on the sequence of Tn6741, as compared to those of other related transposons. Interestingly, although the cloned bla CTX-M-3 gene could confer resistance to ceftiofur, cefquinome, ceftriaxone and cefotaxime, one amino acid substitution (Ile-142-Thr) resulted in a significant reduction of resistance to these antimicrobials. Conclusion. This is the first time that bla CTX-M-3 has been identified on a chromosome from a M. morganii isolate. Furthermore, the bla CTX-M-3 gene was located with an IS26 element and bla TEM-1 gene on a novel IS26-flanked composite transposon, Tn6741, suggesting that Tn6741 might act as a reservoir for the bla CTX-M-3 and bla TEM-1 genes and may become an important vehicle for their dissemination among M. morganii .

Evaluation of the rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test for rapid colistin resistance detection in lactose non-fermenting Gram-negative bacteria

2021 ◽  
Vol 70 (6) ◽  
Author(s):  
Hyunsul Jung ◽  
Johann D. D. Pitout ◽  
Barend C. Mitton ◽  
Kathy-Anne Strydom ◽  
Chanel Kingsburgh ◽  
...  
Keyword(s):  
Type Species ◽  
Susceptibility Testing ◽  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
Major Error ◽  
Colistin Resistance ◽  
Gram Negative ◽  
Content Type ◽  
Link Type ◽  
Extensively Drug Resistant

Introduction. Colistin is one of the last-resort antibiotics for treating multidrug-resistant (MDR) or extensively drug-resistant (XDR) lactose non-fermenting Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii . Gap Statement. As the rate of colistin resistance is steadily rising, there is a need for rapid and accurate antimicrobial susceptibility testing methods for colistin. The Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test has recently been developed for rapid detection of colistin resistance in P. aeruginosa and A. baumannii . Aim. The present study aimed to evaluate the performance of the Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test in comparison with the reference broth microdilution (BMD) method. Methodology. The Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test was performed using a total of 135 P . aeruginosa (17 colistin-resistant and 118 colistin-susceptible) and 66 A. baumannii isolates (32 colistin-resistant and 34 colistin-susceptible), in comparison with the reference BMD method. Results. The categorical agreement of the Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test with the reference BMD method was 97.5 % with a major error rate of 0 % (0/152) and a very major error (VME) rate of 10.2 %. The VME rate was higher (23.5 %) when calculated separately for P. aeruginosa isolates. The overall sensitivity and specificity were 89.8 and 100 %, respectively. Conclusion. The Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test performed better for A. baumannii than for P. aeruginosa .

scholarly journals Detection of Salmonella enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
M. S. R. Fachmann ◽  
C. Löfström ◽  
J. Hoorfar ◽  
F. Hansen ◽  
J. Christensen ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Real Time ◽  
Rapid Method ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Preparation Method ◽  
Reference Method ◽  
Pork Meat ◽  
Sample Preparation Method ◽  
Content Type

ABSTRACT Salmonella is recognized as one of the most important foodborne bacteria and has wide health and socioeconomic impacts worldwide. Fresh pork meat is one of the main sources of Salmonella, and efficient and fast methods for detection are therefore necessary. Current methods for Salmonella detection in fresh meat usually include >16 h of culture enrichment, in a few cases <12 h, thus requiring at least two working shifts. Here, we report a rapid (<5 h) and high-throughput method for screening of Salmonella in samples from fresh pork meat, consisting of a 3-h enrichment in standard buffered peptone water and a real-time PCR-compatible sample preparation method based on filtration, centrifugation, and enzymatic digestion, followed by fast-cycling real-time PCR detection. The method was validated in an unpaired comparative study against the Nordic Committee on Food Analysis (NMKL) reference culture method 187. Pork meat samples (n = 140) were either artificially contaminated with Salmonella at 0, 1 to 10, or 10 to 100 CFU/25 g of meat or naturally contaminated. Cohen's kappa for the degree of agreement between the rapid method and the reference was 0.64, and the relative accuracy, sensitivity, and specificity for the rapid method were 81.4, 95.1, and 97.9%, respectively. The 50% limit of detections (LOD50s) were 8.8 CFU/25 g for the rapid method and 7.7 CFU/25 g for the reference method. Implementation of this method will enable faster release of Salmonella low-risk meat, providing savings for meat producers, and it will help contribute to improved food safety. IMPORTANCE While the cost of analysis and hands-on time of the presented rapid method were comparable to those of reference culture methods, the fast product release by this method can provide the meat industry with a competitive advantage. Not only will the abattoirs save costs for work hours and cold storage, but consumers and retailers will also benefit from fresher meat with a longer shelf life. Furthermore, the presented sample preparation might be adjusted for application in the detection of other pathogenic bacteria in different sample types.

scholarly journals Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147

2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Carla Rodrigues ◽  
Siddhi Desai ◽  
Virginie Passet ◽  
Devarshi Gajjar ◽  
Sylvain Brisse
Keyword(s):  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Antimicrobial Susceptibility ◽  
Type Species ◽  
Susceptibility Testing ◽  
Multidrug Resistant ◽  
Antimicrobial Susceptibility Testing ◽  
Content Type ◽  
Genetic Elements ◽  
Link Type

The rapid emergence of multidrug-resistant Klebsiella pneumoniae is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected: bla NDM-5 and two copies of bla OXA-181 in the chromosome, and a second copy of bla NDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured bla CTX-M. All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. K. pneumoniae CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal bla NDM-5. Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo–spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

scholarly journals Gram-negative bacilli bacteremia: a 7 year retrospective study in a referral Brazilian tertiary-care teaching hospital

2020 ◽  
Author(s):  
Maria Clara Bisaio Quillici ◽  
Daiane Silva Resende ◽  
Iara Rossi Gonçalves ◽  
Sabrina Royer ◽  
Sebastiana Silva Sabino ◽  
...  
Keyword(s):  
Septic Shock ◽  
Antibiotic Therapy ◽  
Bloodstream Infection ◽  
Teaching Hospital ◽  
Type Species ◽  
Bloodstream Infections ◽  
Gram Negative ◽  
Content Type ◽  
Link Type ◽  
Initial Antibiotic Therapy

Introduction. Bloodstream infection is one of the most frequent and challenging hospital-acquired infections and it is associated with high morbidity, mortality and additional use of healthcare resources. Hypothesis/Gap Statement: Bloodstream infections have consequences for the patient, such as the evolution to mortality and inappropriate empirical antibiotic prescription, especially when caused by multidrug-resistant Gram-negative bacilli. Objective. To assess the impact of bloodstream infection and the status of multidrug resistance (MDR) in the evolution of patients who received inappropriate initial antibiotic therapy. Methods. A retrospective surveillance was conducted on nosocomial bloodstream infections caused by Gram-negative bacilli (GNB) from January 2012 to December 2018 in an adult intensive care unit of a Brazilian tertiary teaching hospital. Results. We identified 270 patients with GNB nosocomial bacteremia. Non-survivors were older (with an average age of 58.8 years vs 46.9 years, P=<0.0001), presented more severe illnesses, were immunosuppressed (73.7 vs 37.6%, P=<0.0001), were more likely to have septic shock (55.8 vs 22.4%, P=<0.0001) and had an increased usage of mechanical ventilators (98.6 vs 89.6%, P=0.0013) than survivors. In a logistic regression model, inappropriate empirical antibiotic therapy was not an independent predictor of mortality, different from mechanical ventilator (P=<0.0001; OR=28.0; 95% CI=6.3–123.6), septic shock (P=0.0051; OR=2.5; 95% CI=1.3–4.9) and immunosuppression (P=0.0066; OR=2.6; 95% CI=1.3–5.2). In contrast, in a separate model, MDR was strongly associated with the prescription of inappropriate initial antibiotic therapy (P=0.0030; OR=5.3; 95% CI=1.7–16.1). The main isolated pathogens were Acinetobacter baumannii (23.6 %) and Klebsiella pneumoniae (18.7 %). The frequency of MDR organisms was high (63.7 %), especially among non-fermenting bacilli (60.9 %), highlighting A. baumannii (81.6 %) and Pseudomonas aeruginosa (41.8 %). Conclusion. Illness severity (septic shock and immunosuppression) and mechanical ventilation were identified as predictors of mortality. Additionally, MDR was a major determinant of inappropriate antibiotic empirical therapy, but not associated with mortality, and both characteristics were not statistically associated with death.

To Eliminate Curtain Effect of FIB TEM Samples by a Combination of Sample Dicing and Backside Milling

2014 ◽  
Author(s):  
Jian-Shing Luo ◽  
Hsiu Ting Lee
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Low Cost ◽  
Ion Beam ◽  
Sample Preparation Method ◽  
Site Specific ◽  
Dual Beam ◽  
Transmission Electron

Abstract Several methods are used to invert samples 180 deg in a dual beam focused ion beam (FIB) system for backside milling by a specific in-situ lift out system or stages. However, most of those methods occupied too much time on FIB systems or requires a specific in-situ lift out system. This paper provides a novel transmission electron microscopy (TEM) sample preparation method to eliminate the curtain effect completely by a combination of backside milling and sample dicing with low cost and less FIB time. The procedures of the TEM pre-thinned sample preparation method using a combination of sample dicing and backside milling are described step by step. From the analysis results, the method has applied successfully to eliminate the curtain effect of dual beam FIB TEM samples for both random and site specific addresses.

Cross-Section Sample Preparation Method for Imaging Dopant Related Anomalies Using Scanning Probe Microscopy Techniques

2014 ◽  
Author(s):  
Swaminathan Subramanian ◽  
Khiem Ly ◽  
Tony Chrastecky
Keyword(s):  
Sample Preparation ◽  
Failure Analysis ◽  
Cross Section ◽  
Scanning Probe Microscopy ◽  
Preparation Method ◽  
Fault Isolation ◽  
Scanning Probe ◽  
Sample Preparation Method ◽  
Probe Microscopy ◽  
Section Sample

Abstract Visualization of dopant related anomalies in integrated circuits is extremely challenging. Cleaving of the die may not be possible in practical failure analysis situations that require extensive electrical fault isolation, where the failing die can be submitted of scanning probe microscopy analysis in various states such as partially depackaged die, backside thinned die, and so on. In advanced technologies, the circuit orientation in the wafer may not align with preferred crystallographic direction for cleaving the silicon or other substrates. In order to overcome these issues, a focused ion beam lift-out based approach for site-specific cross-section sample preparation is developed in this work. A directional mechanical polishing procedure to produce smooth damage-free surface for junction profiling is also implemented. Two failure analysis applications of the sample preparation method to visualize junction anomalies using scanning microwave microscopy are also discussed.

scholarly journals Application of the Dehydration Homogeneous Liquid–Liquid Extraction (DHLLE) Sample Preparation Method for Fingerprinting of Honey Volatiles

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2277
Author(s):  
Piotr M. Kuś ◽  
Igor Jerković
Keyword(s):  
Carboxylic Acid ◽  
Sample Preparation ◽  
Preparation Method ◽  
Liquid Extraction ◽  
Sample Preparation Method ◽  
Phenyllactic Acid ◽  
Homogeneous Liquid ◽  
Liquid Liquid Extraction ◽  
Wide Range ◽  
Chemical Groups

Recently, we proposed a new sample preparation method involving reduced solvent and sample usage, based on dehydration homogeneous liquid–liquid extraction (DHLLE) for the screening of volatiles and semi-volatiles from honey. In the present research, the method was applied to a wide range of honeys (21 different representative unifloral samples) to determine its suitability for detecting characteristic honey compounds from different chemical classes. GC-FID/MS disclosed 130 compounds from different structural and chemical groups. The DHLLE method allowed the extraction and identification of a wide range of previously reported specific and nonspecific marker compounds belonging to different chemical groups (including monoterpenes, norisoprenoids, benzene derivatives, or nitrogen compounds). For example, DHLLE allowed the detection of cornflower honey chemical markers: 3-oxo-retro-α-ionols, 3,4-dihydro-3-oxoedulan, phenyllactic acid; coffee honey markers: theobromine and caffeine; linden honey markers: 4-isopropenylcyclohexa-1,3-diene-1-carboxylic acid and 4-(2-hydroxy-2-propanyl)cyclohexa-1,3-diene-1-carboxylic acid, as well as furan derivatives from buckwheat honey. The obtained results were comparable with the previously reported data on markers of various honey varieties. Considering the application of much lower volumes of very common reagents, DHLLE may provide economical and ecological advantages as an alternative sample preparation method for routine purposes.

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