Biology subjects

Kluytmans, J. A. J. W.

Publications and source records attributed to Kluytmans, J. A. J. W..

5 recordsLinked to original sources

bioRxiv2020

Genetic diversity of blaKPC-gene-containing IncF plasmids from epidemiologically related and unrelated Enterobacteriaceae

BackgroundLimited information is available on whether blaKPC-containing plasmids from isolates in a hospital outbreak can be differentiated from epidemiologically unrelated blaKPC-containing plasmids based on sequence data. ObjectiveThis study aimed to evaluate the performance of three approaches to distinguish epidemiologically related from unrelated blaKPC-containing IncF plasmids. MethodEpidemiologically related isolates, were short- and long-read whole genome sequenced on an Illumina MiSeq and MinION sequencer. A hybrid assembly was performed and plasmid sequences were extracted from the assembly graph. Epidemiologically unrelated plasmid sequences were extracted from the GenBank. Pairwise comparisons were performed of epidemiologically related and unrelated plasmids based on SNP differences using snippy, phylogenetic distance using Roary and using a similarity index that penalizes size differences between plasmids (Stoesser-index). The percentage of pairwise comparisons misclassified as genetically related or as clonally unrelated was determined using different genetic thresholds for genetic relatedness for all three comparison methods. ResultsDespite the median number of SNP differences, Roary phylogenetic distance, and Stoesser-index differed between the epidemiologically related and unrelated plasmids, the range of differences overlapped between the two comparison groups for all three comparison methods. When using a genetic similarity threshold that classified 100% of epidemiologically related plasmid pairs as genetically related, the percentages of plasmids misclassified as epidemiologically related ranged from 6.7% (Roary) to 20.8% (Stoesser-index). DiscussionAlthough epidemiologically related plasmids can be distinguished from unrelated plasmids based on genetic similarity, epidemiologically related and unrelated blaKPC-containing IncF plasmids show a high degree of sequence similarity. The phylogenetic distance as determined using Roary showed the highest degree of discriminatory power between the epidemiologically related and unrelated plasmids. Impact statementAccurately distinguishing epidemiologically related from unrelated plasmids is essential to detect nosocomial plasmid transmission in outbreaks. However, limited information is available on whether blaKPC-containing plasmids from isolates in a hospital outbreak can be differentiated from epidemiologically unrelated blaKPC-containing plasmids based on sequence data. This study aimed to evaluate the performance of three approaches to distinguish epidemiologically related from unrelated blaKPC-containing IncF plasmids. Pairwise comparisons were performed of epidemiologically related and unrelated plasmids based on SNP differences using snippy, phylogenetic distance using Roary and using a similarity index that penalizes size differences between plasmids (Stoesser-index). Based on our results, epidemiologically related plasmids can be distinguished from unrelated plasmids based on genetic similarity. Despite this, epidemiologically related and unrelated blaKPC-containing IncF plasmids show a high degree of sequence similarity and judgements on the horizontal transfer of these plasmids during hospital outbreaks based on genetic identity should be made with caution. The phylogenetic distance determined using Roary showed the highest discriminatory power between the epidemiologically related and unrelated plasmids. Data summaryShort-and long-read sequence data of the epidemiologically related Enterobacteriaceae isolates included in this study are available from the publicly available European Nucleotide Archive of the European Bioinformatics Institute under study accession number: PRJEB41009. The authors confirm that all supporting data have been provided within the article and through the supplementary data files.

microbiology↗

bioRxiv2019

Detection of extended-spectrum beta-lactamase (ESBL) genes and plasmid replicons in Enterobacteriaceae using PlasmidSPAdes assembly of short-read sequence data

INTRODUCTIONKnowledge of the epidemiology of plasmids is essential for understanding the evolution and spread of antimicrobial resistance. PlasmidSPAdes attempts to reconstruct plasmids using short-read sequence data. Accurate detection of extended-spectrum beta-lactamase (ESBL) genes and plasmid replicon genes is a prerequisite for the use of plasmid assembly tools to investigate the role of plasmids in the spread and evolution of ESBL production in Enterobacteriaceae. This study evaluated the performance of PlasmidSPAdes plasmid assembly for Enterobacteriaceae in terms of detection of ESBL-encoding genes, plasmid replicons and chromosomal genes, and assessed the effect of k-mer size. METHODSShort-read sequence data of 59 ESBL-producing Enterobacteriaceae were assembled with PlasmidSPAdes using different k-mer sizes (21, 33, 55, 77, 99 and 127). For every k-mer size, the presence of ESBL genes, plasmid replicons and a selection of chromosomal genes in the plasmid assembly was determined. RESULTSOut of 241 plasmid replicons and 66 ESBL genes detected by whole-genome assemblies, 213 plasmid replicons (88%; 95% Confidence Interval (CI): 83.9-91.9) and 43 ESBL genes (65%; 95%CI: 53.1-75.6) were detected in the plasmid assemblies obtained by PlasmidSPAdes. For most ESBL genes (83.3%) and plasmid replicons (72.0%), detection results did not differ between the k-mer sizes used in the plasmid assembly. No optimal k-mer size could be defined for the number of ESBL genes and plasmid replicons detected. For most isolates, the number of chromosomal genes detected in the plasmid assemblies decreased with increasing k-mer size. CONCLUSIONBased on our findings, PlasmidSPAdes is not a suitable plasmid assembly tool for short-read sequence data of ESBL-encoding plasmids of Enterobacteriaceae.

microbiology↗

bioRxiv2019

Detection of AmpC β-lactamases in Escherichia coli using different screening agars

The aim of this study was to determine the performance of both cefotaxime and ceftazidime containing agars on the specificity and sensitivity for chromosomal AmpC-hyperproducing and plasmid AmpC harboring Escherichia coli compared to ESBL-producing E. coli and E. coli without ESBL, pAmpC or cAmpC hyperproduction. Second, we evaluated the influence of adding cefoxitin to these agars for detection of both chromosomal AmpC-hyperproducing and plasmid AmpC harboring E. coli.\n\nFour different homemade screening agars with cefotaxime (1mg/L), ceftazidime (1mg/L), cefotaxime (1mg/L) with cefoxitin (8mg/L), and ceftazidime (1mg/L) with cefoxitin (8mg/L) were compared to each other for the identification of AmpC producing E. coli. A total of 40 isolates with plasmid encoded AmpC {beta}-lactamases, 40 isolates with alterations in the promoter/attenuator region of the AmpC gene leading to hyperproduction of the {beta}-lactamase, 40 isolates with ESBL genes and 39 isolates lacking both a AmpC and ESBL genotype were used to test the four agars.\n\nThe sensitivity and specificity were 100% (95% confidence interval (95% CI) 96.1% to 100%) and 48.1% (95% CI 38.6%-60.2%), respectively, for the cefotaxime agar; 100% (95% CI 96.1% to 100%) and 49.41% (95% CI 39.8%-61.4%), respectively, for the ceftazidime agar; 96.3% (95% CI 89.1% to 99.2%) and 77.2% (95% CI 66.7%-85.2%) respectively, for the cefotaxime with cefoxitin agar; 98.8% (95% CI) 92.6% to 99.6%) and 81.0% (95% CI 70.9%-88.3%) respectively, for the ceftazidime agar with cefoxitin. The main reason for false-positive results were ESBL-harboring strains that grew on various agars; therefore, the specificity of each agar reported here was influenced mainly by the proportion of ESBL isolates tested. In conclusion addition of cefoxitin to cefotaxime and ceftazidime containing agars had little influence on sensitivity, but increased specificity for the detection of AmpC in E. coli.

microbiology↗

bioRxiv2019

Extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing Escherichia coli isolates causing bacteremia in the Netherlands (2014 - 2016) differ in ST distribution, antimicrobial resistance gene and virulence gene content

BackgroundKnowledge on the molecular epidemiology of Escherichia coli causing E. coli bacteremia (ECB) in the Netherlands is mostly based on extended-spectrum beta-lactamase-producing E. coli (ESBL-Ec). We determined differences in clonality and resistance and virulence gene (VG) content between non-ESBL-producing E. coli (non-ESBL-Ec) and ESBL-Ec blood isolates with different epidemiological characteristics.\n\nMaterials/methodsA random selection of non-ESBL-Ec isolates as well as all available ESBL-Ec blood isolates was obtained from two Dutch hospitals between 2014 and 2016. Whole genome sequencing was performed to infer sequence types (STs), serotypes, acquired antibiotic resistance genes and VG scores, based on presence of 49 predefined putative pathogenic VG.\n\nResultsST73 was most prevalent among the 212 non-ESBL-Ec (N=26, 12.3%) and ST131 among the 69 ESBL-Ec (N=30, 43.5%). Prevalence of ST131 among non-ESBL-Ec was 10.4% (N=22, P value < 0.001 compared to ESBL-Ec). O25:H4 was the most common serotype in both non-ESBL-Ec and ESBL-Ec. Median acquired resistance gene counts were 1 (IQR 1 - 6) and 7 (IQR 4 - 9) for non-ESBL-Ec and ESBL-Ec, respectively (P value < 0.001). Among non-ESBL-Ec, acquired resistance gene count was highest among blood isolates from a primary gastro-intestinal focus (median 4, IQR 1 - 8). Median VG scores were 13 (IQR 9 - 20) and 12 (IQR 8 - 14) for non-ESBL-Ec and ESBL-Ec, respectively (P value = 0.002). VG scores among non-ESBL-Ec from a primary urinary focus (median 15, IQR 11 - 21) were higher compared to non-ESBL-Ec from a primary gastro-intestinal (median 10, IQR 6 - 13) or hepatic-biliary focus (median 11, IQR 5 - 18) (P values = 0.007 and 0.036, respectively). VG content varied between different E. coli STs.\n\nConclusionsNon-ESBL-Ec and ESBL-Ec blood isolates from two Dutch hospitals differed in clonal distribution, resistance gene and VG content. Also, resistance gene and VG content differed between non-ESBL-Ec from different primary foci of ECB.

microbiology↗

bioRxiv2017

Diagnostic Prediction Tools For Bacteraemia Caused By 3rd Generation Cephalosporin-Resistant Enterobacteriaceae In Suspected Bacterial Infections: A Nested Case-Control Study

ObjectivesCurrent guidelines for empirical antibiotic treatment poorly predict the presence of 3rd generation cephalosporin resistant Enterobacteriaceae (3GC-R EB) as a cause of infection, thereby increasing unnecessary carbapenem use. We aimed to develop diagnostic scoring systems to better predict the presence of 3GC-R EB as a cause of bacteraemia.\n\nMethodsA retrospective nested case-control study was performed that included patients [≥]18 years in whom blood cultures were obtained and intravenous antibiotics were initiated. Each patient with 3GC-R EB bacteraemia was matched to four control infection episodes within the same hospital, based on blood culture date and onset location (community or hospital). Starting from 32 described clinical risk factors at infection onset, selection strategies were used to derive scoring systems for the probability of community- and hospital-onset 3GC-R EB bacteraemia.\n\nResults3GC-R EB bacteraemia occurred in 90 of 22,506 (0.4%) community-onset and in 82 of 8,110 (1.0%) hospital-onset infections, and these cases were matched to 360 community-onset and 328 hospital-onset control episodes, respectively. The derived community-onset and hospital-onset scoring system consisted of 6 and 9 predictors, respectively, with c-statistics of 0.807 (95% confidence interval 0.756-0.855) and 0.842 (0.794-0.887). With selected score cutoffs, the models identified 3GC-R EB bacteraemia with equal sensitivity as existing guidelines, but reduced the proportion of patients classified as at risk for 3GC-R EB bacteraemia (i.e. eligible for empiric carbapenem therapy) with 40% in patients with community-onset and 49% in patients with hospital-onset infection.\n\nConclusionsThese prediction rules for 3GC-R EB bacteraemia may reduce unnecessary empiric carbapenem use.

epidemiology↗