Biology subjects

Verweij, J. J.

Publications and source records attributed to Verweij, J. J..

3 recordsLinked to original sources

bioRxiv2024

The assessment of single nucleotide polymorphisms in the ss-tubulin genes in human soil-transmitted helminths exposed to different pressure with benzimidazole drugs

BackgroundWe aimed to gain insights into the role of known single nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 of the {beta}-tubulin gene as markers for possible benzimidazole resistance in human soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus and Ancylostsoma duodenale). MethodsFirstly, we determined the analytical performance of our PCR/pyrosequencing assays. Secondly, we applied them on stool samples collected during clinical trials in Ethiopia, Lao PDR, and Pemba Island (Tanzania) to assess any associations between the presence/ratio of mutant (MT): wild type (WT) SNPs and drug pressure history, individual drug response and time of sampling (baseline vs. follow-up sample). Principal findingsOverall, the limit of blank of our in-house PCR/pyrosequencing assays to detect MT SNPs was non-zero ([~]3.5%), and hence the limit of detection for MT SNPs was relatively high (2% - 7%). The assays systematically overestimated the true underlying ratio of MT:WT SNPS within sample, but we derived functions for more accurate estimates. The assays were more precise when the ratio MT:WT SNPs was high (>5%). No PCR amplicon was observed in 25% of the samples subjected to PCR. In the remaining samples, the presence of MT SNPs in codon 200 was detected in half of the analysed Trichuris samples, the proportion of the analysed samples containing MT SNPs did not exceed 14% for all other codons and STH species. Associations between drug pressure history, individual drug response and time of sampling, were not consistent across all codons and STHs. ConclusionWe could not provide compelling evidence for the role of the known SNPs in the {beta}-tubulin gene as markers for benzimidazole resistance. Our study also highlights that there is a need to assess the diagnostic performance of any assays in order to readily interpret and compare results. Further research should therefore also focus on genes other than the {beta}-tubulin genes. Author summaryAlthough large-sale deworming programs are reducing the morbidity caused by intestinal worms, widespread treatment of large populations for a long period of time may trigger drug resistance. An early detection of DNA mutations that may give rise to resistant worm population is therefore important. We evaluated the analytical performance of in-house assays to detect DNA mutations that are known to cause resistant intestinal worms of animals. Subsequently, we applied these assays on stool samples to verify (i) whether the mutations are more prevalent in areas were large proportions of children have been dewormed for a longer period, (ii) a poor individual drug response can be explained by higher frequency of the mutations. Our results indicate that comprehensive evaluation of the analytical performance of the genotyping tests was required to readily interpret the results. We did not find any compelling evidence that the presence of mutations was associated with either drug pressure or poor individual drug responses. This suggests that it is warranted to explore other mutations than those documented in animal worms.

molecular biology↗

bioRxiv2020

Genome-wide analysis in Escherichia coli unravels an unprecedented level of genetic homoplasy associated with cefotaxime resistance.

ABSTRACTCefotaxime (CTX) is a commonly used third-generation cephalosporin (3GC) to treat infections caused by Escherichia coli. Two genetic mechanisms have been associated with 3GC resistance in E. coli. The first is the conjugative transfer of a plasmid harboring antibiotic resistance genes. The second is the introduction of mutations in the promoter region of the ampC β-lactamase gene that cause chromosomal-encoded β-lactamase hyperproduction. A wide variety of promoter mutations related to AmpC hyperproduction have been described. However, their link to a specific 3GC such as CTX resistance has not been reported. Here, we measured CTX MICs in 172 cefoxitin resistant E. coli isolates and performed genome-wide analysis of homoplastic mutations associated with CTX resistance by comparing Illumina whole-genome sequencing data of all isolates to a PacBio tailored-made reference chromosome. We mapped the mutations on the reference chromosome and determined their occurrence in the phylogeny, revealing extreme homoplasy at the −42 position of the ampC promoter. The 24 occurrences of a “T” at the −42 position rather than the wild type “C”, resulted from 18 independent C>T mutations in 5 phylogroups. The −42 C>T mutation was only observed in E. coli lacking a plasmid-encoded ampC gene. The association of the −42 C>T mutation with CTX resistance was confirmed to be significant (FDR < 0.05). To conclude, genome-wide analysis of homoplasy in combination with CTX resistance identifies the −42 C>T mutation of the ampC promotor as significantly associated with CTX resistance and underline the role of recurrent mutations in the spread of antibiotics resistance.Impact Statement In the past decades, the worldwide spread of extended spectrum beta-lactamases (ESBLs) has led to a substantial increase in the prevalence of resistant common pathogens, thereby restricting available treatment options. Although acquired resistance genes, e.g. ESBLs, get most attention, chromosome-encoded resistance mechanisms may play an important role as well. In E. coli chromosome-encoded β-lactam resistance can be caused by alterations in the promoter region of the ampC gene. To improve our understanding of how frequently these alterations occur, a comprehensive interpretation of the evolution of these mutations is essential. This study is the first to apply genome-wide homoplasy analysis to better perceive adaptation of the E. coli genome to antibiotics. Thereby, this study grants insights into how chromosomal-encoded antibiotic resistance evolves and, by combining genome-wide association studies with homoplasy analyses, provides potential strategies for future association studies into the causes of antibiotics resistance.Data summary All data is available under BioProject: PRJNA592140. Raw Illumina sequencing data and metadata of all 171 E. coli isolates used in this study is available from the Sequence Read Archive database under accession no. SAMN15052485 to SAMN15052655. Full reference chromosome of ampC_0069 is available via GenBank accession no. CP046396.1 and NCBI Reference Sequence: NZ_CP046396.1.Competing Interest StatementThe authors have declared no competing interest.Abbreviations3GCthird-generation cephalosporincampCchromosomal-mediated ampCCATcomputerized adaptive testingCTXcefotaximeDNADeoxyribonucleic acidEHECenterohemorrhagic Escherichia ColiESBLextended-spectrum β-lactamasesFOXcefoxitingDNAgenomic DNAMICsminimal inhibitory concentrationsMLSTmultilocus sequence typingpampCplasmid-mediated ampCPGpeptidoglycanqRT-PCRquantitative reverse transciptase polymerase chain reactionSMRTSingle-molecule real-time sequencingSNPsingle-nucleotide polymorphismSTsequence typeUPECuropathogene Escherichia ColiWGSwhole genome sequencingView Full Text

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↗