Biology subjects

Cuenod, A.

Publications and source records attributed to Cuenod, A..

10 recordsLinked to original sources

bioRxiv2025

Ciprofloxacin resistance in Klebsiella pneumoniae: phenotype prediction from genotype and global distribution of resistance determinants

BACKGROUNDCiprofloxacin resistant Klebsiella pneumoniae is common or emerging in many geographies, and knowledge of local resistance rates is important for empirical therapy. Whilst there are known K. pneumoniae ciprofloxacin resistance determinants, there is a lack of systematic data on the effect of determinants, alone and in combination, and there are no publicly accessible tools for predicting resistance from whole genome sequence data. METHODSThe KlebNET-GSP AMR Genotype-Phenotype Group aggregated a matched genotype-phenotype dataset of n=12,167 K. pneumoniae species complex (KpSC) isolates from 27 countries between 2001-2021. We developed a rules-based classifier to predict ciprofloxacin resistance by categorizing the number of quinolone resistance determining regions mutations in gyrA and parC, the number of plasmid-mediated quinolone resistance genes, and the presence/absence of aac(6')-Ib-cr (which can acetylate ciprofloxacin). Predictive performance was assessed using the discovery dataset, for which we re-phenotyped discrepant isolates; and validated using externally contributed datasets (n=7,030 KpSC isolates). RESULTSThe rules-based classifier predicted R vs S/I with categorical agreement, sensitivity, and specificity >96%, and major/very major error rates <4%. Performance was similar across diverse KpSC sources (human, animal, other), species, and intra-species lineages. External validation of the classifier yielded overall 93.12% categorical agreement [95% confidence interval (CI), 92.50-93.74%], 8.65% major errors [95% CI, 7.34-9.97%], and 6.20% very major errors [95% CI, 5.51-6.90%]. We implemented the classifier in Kleborate, a command-line tool that is integrated into the Pathogenwatch web platform. Using this to assess the global distribution of ciprofloxacin resistance determinants in KpSC genomes available in Pathogenwatch (n=31,319, from 109 countries between years 2000-2023), we observed a significant positive association between national quinolone consumption rates and predicted ciprofloxacin resistance (R2=0.20, p=0.004). CONCLUSIONS Ciprofloxacin resistance phenotypes can be reasonably predicted from genotypes, which is sufficient for informing surveillance. However, unexplained resistance remains and accuracy is insufficient for clinical applications. We demonstrate the value of aggregating genotype-phenotype data to explore resistance mechanisms and develop predictors, but highlight complexities in combining phenotype data from different assays and standards.

genomics↗

bioRxiv2025

The natural diversity of E. coli transporter-dependent capsules

Serotyping of bacteria using genomic information (in silico serotyping) has increasingly replaced serology. However, the E. coli capsule serotyping system has been largely abandoned since the 1990s, leaving gaps in our knowledge of capsule genetics, diversity, distribution, and epidemiology. To address this, we established a definitive genotype-serotype map for 35 serologically identified and structurally characterized transporter-dependent capsules. We then surveyed >37,000 E. coli genomes, cataloging 85 transporter-dependent capsule types (K-types), including 55 novel ones. We leveraged this catalog to develop an in silico serotyping tool, kTYPr, and applied it to curated sets of >25,000 E. coli genomes and metagenome-assembled genomes spanning diverse environmental and clinical sources. We found novel K-types enriched in under-sampled environments and associated with E. coli disease. This research expands our understanding of E. coli surface structures, supporting efforts for precision targeting with phage therapy or vaccines.

microbiology↗

bioRxiv2024

Unlocking hidden phage diversity to complete the BASEL collection for systematic exploration of phage-host interactions

Research on bacteriophages, the viruses infecting bacteria, has fueled the development of modern molecular biology and inspired their therapeutic application to combat bacterial multidrug resistance. However, most work has so far focused on a few model phages which impedes direct applications of these findings in clinics and suggests that a vast potential of powerful molecular biology has remained untapped. We have therefore recently composed the BASEL collection of Escherichia coli phages (BActeriophage SElection for your Laboratory) which made a relevant diversity of phages infecting the E. coli K-12 laboratory strain accessible to the community. These phages are widely used, but their assorted diversity had remained limited by the E. coli K-12 host. We have therefore now genetically overcome the two major limitations of E. coli K-12, its lack of O-antigen glycans and the presence of resident bacterial immunity. Restoring O-antigen expression resulted in the isolation of diverse additional viral groups like Kagunavirus, Nonanavirus, Gordonclarkvirinae, and Gamaleyavirus, while eliminating all known antiviral defenses of E. coli K-12 additionally enabled us to isolate phages of Wifcevirus genus. Even though some of these viral groups appear to be common in nature, no phages from any of them had previously been isolated using E. coli laboratory strains and they had thus remained largely understudied. Overall, 37 new phage isolates have been added to complete the BASEL collection. These phages were deeply characterized genomically and phenotypically with regard to host receptors, sensitivity to antiviral defense systems, and host range. These results highlighted dominant roles of the O-antigen barrier for viral host recognition and of restriction-modification systems in bacterial immunity. We anticipate that the completed BASEL collection will propel research on phage-host interactions and their molecular mechanisms, deepening our understanding of viral ecology and fostering innovations in biotechnology and antimicrobial therapy.

microbiology↗

bioRxiv2024

Prevalent chromosome fusion in Vibrio cholerae O1

Two circular chromosomes are a defining feature of the family Vibrionaceae, including the pathogen Vibrio cholerae, with rare reports of isolates with a single, fused chromosome. Here we report chromosome fusions in clinical V. cholerae O1 isolates, including several independent fusion events stable enough to be transmitted between patients within a household. Fusion occurs in a 12 kilobase-pair homologous sequence shared between the two chromosomes, which may lead to reversible chromosomal fusion.

microbiology↗

bioRxiv2024

Diversity, functional classification and genotyping of SHV β-lactamases in Klebsiella pneumoniae

Interpreting phenotypes of blaSHV alleles in Klebsiella pneumoniae genomes is complex. While all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal blaSHV alleles or additional plasmid-borne blaSHV alleles that have extended-spectrum {beta}-lactamase (ESBL) activity and/or {beta}-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/amino acid changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases (e.g., NCBIs Reference Gene Catalog and the {beta}-lactamase database (BLDB)) over the specific functionality of individual SHV protein variants. Therefore, identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific amino-acid substitutions. We then systematically assigned SHV alleles to functional classes (wildtype, ESBL, BLI-resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with current assignments in NCBIs Reference Gene Catalog and/or BLDB (21 to wildtype, 12 to ESBL, 4 to BLI-resistant). Phylogenetic and comparative genomic analyses support that; i) SHV-1 (encoded by blaSHV-1) is the ancestral chromosomal variant; ii) ESBL and BLI-resistant variants have evolved multiple times through parallel substitution mutations; iii) ESBL variants are mostly mobilised to plasmids; iv) BLI-resistant variants mostly result from mutations in chromosomal blaSHV. We used matched genome-phenotype data from the KlebNET-GSP Genotype-Phenotype Group to identify 3,999 K. pneumoniae isolates carrying one or more blaSHV alleles but no other acquired {beta}-lactamases, with which we assessed genotype-phenotype relationships for blaSHV. This collection includes human, animal, and environmental isolates collected between 2001 to 2021 from 24 countries across six continents. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, while most omega-loop substitutions do not. Our data also provide direct support for wildtype assignment of 67 protein variants, including eight that were noted in public databases as ESBL. We reclassified these eight variants as wildtype, because they lack ESBL-associated mutations, and our phenotype data support susceptibility to 3GCs (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164, SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae from genome assemblies), whereby known and novel blaSHV alleles are classified based on causative mutations. Kleborate v2.4.1 was also updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve understanding of resistance mechanisms. Impact statementSince every K. pneumoniae genome has an intrinsic SHV {beta}-lactamase and may also carry additional mobile forms, the correct interpretation of blaSHV genes detected in genome data can be challenging and can lead to K. pneumoniae being misclassified as ESBL-producing. Here, we use matched K. pneumoniae genome and drug susceptibility data contributed from dozens of studies, together with systematic literature review of experimental evidence, to improve our understanding of blaSHV allele variation and mapping of genotype to phenotype. This study shows the value of coordinated data sharing, in this case via the KlebNET-GSP Genotype-Phenotype Group, to improve our understanding of the evolutionary history and functionality of blaSHV genes. The results are captured in an open-source AMR dictionary utilised by the Kleborate genotyping tool, that could easily be incorporated into or used to update other tools and AMR gene databases. This work is part of the wider efforts of the KlebNET-GSP group to develop and support a unified platform tailored for the analysis and interpretation of K. pneumoniae genomes by a wide range of stakeholders. Data summaryBlaSHV allele sequences and class assignments are distributed with Kleborate, v2.4.1, DOI:10.5281/zenodo.10469001. Table S1 provides a summary of blaSHV alleles, including primary accessions, class-modifying mutations, and supporting evidence for class assignments that differ from NCBIs Reference Gene Catalog or BLDB. Whole genome sequence data are publicly available as reads and/or assemblies, individual accessions are given in Table S2; corresponding genotypes and antibiotic susceptibility phenotypes and measurements are available in Tables S3 and S4, respectively.

genomics↗

bioRxiv2023

Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans

Type IV-A CRISPR-Cas systems are primarily encoded on plasmids and form multi-subunit ribonucleoprotein complexes with unknown biological functions. In contrast to other CRISPR-Cas types, they lack the archetypical CRISPR acquisition module and encode a DinG helicase instead of a nuclease component. Type IV-A3 systems are carried by large conjugative plasmids that often harbor multiple antibiotic-resistance genes. Although their CRISPR array contents suggest a role in inter-plasmid conflicts, this function and the underlying mechanisms have remained unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 CRISPR-Cas system co-opts the type I-E adaptation machinery from its clinical Klebsiella pneumoniae host to update its CRISPR array. Furthermore, we demonstrate that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By targeting plasmid core functions, type IV-A3 can prevent the uptake of incoming plasmids, limit their horizontal transfer, and destabilize co-residing plasmids, altogether supporting type IV-A3s involvement in plasmid competition. Collectively, our findings shed light on the molecular mechanisms and ecological function of type IV-A3 systems and have broad implications for understanding and countering the spread of antibiotic resistance in clinically relevant strains.

microbiology↗

bioRxiv2023

Phage predation and antibiotic exposure are inversely associated with disease severity and shape pathogen genetic diversity in cholera patients

Despite an increasingly detailed picture of the molecular mechanisms of phage-bacterial interactions, we lack an understanding of how these interactions evolve and impact disease within patients. Here we report a year-long, nation-wide study of diarrheal disease patients in Bangladesh. Among cholera patients, we quantified Vibrio cholerae (prey) and its virulent phages (predators) using metagenomics and quantitative PCR, while accounting for antibiotic exposure using quantitative mass spectrometry. Virulent phage (ICP1) and antibiotics suppressed V. cholerae to varying degrees and were inversely associated with severe dehydration depending on resistance mechanisms. In the absence of anti-phage defenses, predation was effective, with a high predator:prey ratio that correlated with increased genetic diversity among the prey. In the presence of anti-phage defenses, predation was ineffective, with a lower predator:prey ratio that correlated with increased genetic diversity among the predators. Phage-bacteria coevolution within patients should therefore be considered in the deployment of phage-based therapies and diagnostics. One Sentence SummaryA survey of cholera patients in Bangladesh identifies phage predation as a biomarker of disease severity and driver of coevolution within patients.

microbiology↗

bioRxiv2023

Ancient methicillin-resistant Staphylococcus aureus: expanding current knowledge using molecular epidemiological characterization of a Swiss legacy collection

Few methicillin-resistant Staphylococcus aureus (MRSA) from the early years of its global emergence have been sequenced. Knowledge about evolutionary factors promoting the success of specific MRSA multi-locus sequence types (MLSTs) remains scarce. We aimed to characterize a legacy MRSA collection isolated from 1965 to 1987 and compare it against publicly available international and local genomes. We accessed 451 ancient (1965-1987) Swiss MRSA isolates, stored in the Culture Collection of Switzerland. We determined phenotypic antimicrobial resistance (AMR) and performed Illumina short-read sequencing on all isolates and long-read sequencing on a selection with Oxford Nanopore Technology. For context, we included 103 publicly available international genomes from 1960 to 1992 and sequenced 1207 modern Swiss MRSA isolates from 2007 to 2022. We analyzed the core genome (cg)MLST and predicted SCCmec cassette types, AMR, and virulence genes. Among the 451 ancient Swiss MRSA isolates, we found 17 sequence types (STs) of which 11 have been previously described. Two STs were novel combinations of known loci and six isolates carried previously unsubmitted MLST alleles, representing five new STs (ST7843, ST7844, ST7837, ST7839, and ST7842). Most isolates (83% 376/451) represented ST247-MRSA-I isolated in the 1960s, followed by ST7844 (6% 25/451), a novel single locus variant (SLV) of ST239. Analysis by cgMLST indicated that isolates belonging to ST7844-MRSA-III cluster within the diversity of ST239-MRSA-IIII. Early MRSA were predominantly from clonal complex (CC) 8. From 1980 to the end of the 20th century we observed that CC22 and CC5 as well as CC8 were present, both locally and internationally. The combined analysis of 1761 ancient and contemporary MRSA isolates across more than 50 years uncovered novel STs and allowed us a glimpse into the lineage flux between Swiss and international MRSA across time.

microbiology↗

bioRxiv2022

Improved MALDI-TOF MS based antimicrobial resistance prediction through hierarchical stratification

MotivationPredicting antimicrobial resistance using MALDI-TOF mass spectrometry based machine learning is a fast-growing field of research. Recent advances in machine learning methods specifically designed for MALDI-TOF mass spectra have outperformed established classification approaches. However, classification performance was observed to have a large standard deviation between different train-test splits. We hypothesise that this variance is caused by the underlying phylogenetic structure between microbial samples, which is implicitly reflected in their MALDI-TOF MS profiles, but not taken into account during the training of a model. ResultsIn this paper, we propose to infer this structure from the dataset--using agglomerative hierarchical clustering--and consider it during the dataset splitting between train and test. We show that incorporating such phylogenetic structure into the antimicrobial resistance prediction scenario leads to an improved classification performance. Average precision was increased from 42.3 to 47.1 for ciprofloxacin resistance prediction in Escherichia coli and from 44.6 to 50.8 for amoxicillin-clavulanic acid resistance prediction in Staphylococcus aureus using a Gaussian process classifier with a MALDI-TOF MS specific kernel. We envision that these results will support the quick and reliable identification of antimicrobial resistances, thus increasing patient well-being and reducing healthcare costs. AvailabilityAll data is available for download and code available as an easy-to-use Python package under https://github.com/BorgwardtLab/maldi_PIKE at branch maldi_stratification. Contactcaroline.weis@bsse.ethz.ch, karsten.borgwardt@bsse.ethz.ch Supplementary informationSupplementary information at the end of document.

bioinformatics↗

bioRxiv2020

Direct Antimicrobial Resistance Prediction from MALDI-TOF mass spectra profile in clinical isolates through Machine Learning

Early administration of effective antimicrobial treatments is critical for the outcome of infections. Antimicrobial resistance testing enables the selection of optimal antibiotic treatments, but current culture-based techniques take up to 72 hours. We have developed a novel machine learning approach to predict antimicrobial resistance directly from MALDI-TOF mass spectra profiles of clinical samples. We trained calibrated classifiers on a newly-created publicly available database of mass spectra profiles from clinically most relevant isolates with linked antimicrobial susceptibility phenotypes. The dataset combines more than 300,000 mass spectra with more than 750,000 antimicrobial resistance phenotypes from four medical institutions. Validation against a panel of clinically important pathogens, including Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, resulting in AUROC values of 0.8, 0.74, and 0.74 respectively, demonstrated the potential of using machine learning to substantially accelerate antimicrobial resistance determination and change of clinical management. Furthermore, a retrospective clinical case study found that implementation of this approach would have resulted in a beneficial change in the clinical treatment in 88% (8/9) of cases. MALDI-TOF mass spectra based machine learning may thus be an important new tool for antibiotic stewardship.

microbiology↗