Biology subjects

Giani, T.

Publications and source records attributed to Giani, T..

5 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

Neighbour Typing Using Long Read Sequencing Provides Rapid Prediction of Sequence Type and Antimicrobial Susceptibility of Klebsiella pneumoniae

The rapid genome-based diagnostic approach of long read sequencing coupled with neighbor typing offers the potential to improve empiric treatment of infection. However, this approach is still in development, and clinical validation is needed to support its use. In this study, we present an assessment of a neighbour typing method (RASE - resistance associated sequence elements) to predict lineage or sequence type (ST) and antimicrobial susceptibility in real time for Klebsiella pneumoniae sensu lato. We analysed the initial reads generated during the early phase of long read sequencing from pure culture (n=99), mock communities (n=20) and metagenomic samples (n=20). RASE accurately identified 69.7% and 70% of STs in pure culture and metagenomes, respectively, and identified the STs of the isolates representing the highest proportion in mock communities. Regarding antimicrobial susceptibility prediction, the probability of susceptibility increased to 72% (95% CI 63%-80%) across all tested antibiotics, when RASE predicted susceptibility, and decreased probability of susceptibility to 8.9% (95% CI 6.4%-9.6%) when was indicative of a resistant phenotype. Our study confirmed that genomic neighbor typing in K. pneumoniae sensu lato is capable of providing informative predictions of ST and antibiotic susceptibility in less than ten minutes (after the start of sequencing) with 200-500 reads. IMPORTANCEThe growing burden of antimicrobial resistance is leading to high rates of mortality and morbidity worldwide. This situation has made the selection of empirical antibiotic therapy challenging, due to the risk of treatment failure and the overuse of last-resort antibiotics. The development of new sequencing technologies is helping to reduce the waiting time for a microbiological diagnosis, providing information in the early phase of bacterial infections, which could help improve clinical outcomes in a time of rising antimicrobial resistance. In this context, we assessed the performance of RASE (resistance associated sequence elements) in Klebsiella pneumoniae, an opportunistic pathogen frequently associated with nosocomial infections, which can rapidly acquire antibiotic resistance genes. Thus, in our study we provide insights that may aid in the validation of RASE for clinical use.

genomics↗

bioRxiv2022

Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. In a murine pneumonia model, KP mutants harbouring L3 insertions have a competitive disadvantage relative to a strain expressing wild-type OmpK36 in the absence of antibiotics. This explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies. Author summaryRapidly rising rates of antibiotic resistance among Klebsiella pneumoniae (KP) necessitate a comprehensive understanding of the diversity, spread and clinical impact of resistance mutations. In KP, mutations in outer membrane porins play an important role in mediating resistance to carbapenems, a key class of antibiotics. Here we show that resistance mutations in the extracellular loop 3 (L3) region of the OmpK36 porin are found at high prevalence among clinical genomes and we characterise their diversity and impact on resistance and virulence. They include amino acid insertions of Aspartate (D), Glycine-Aspartate (GD) and Threonine-Aspartate (TD), which act by decreasing the pore size and restricting entry of carbapenems into the bacterial cell. We show that these L3 insertions are associated with large clonal expansions of resistant lineages and impose only a low fitness cost. Critically, strains harbouring L3 insertions remain susceptible to novel drugs, including beta lactam-beta lactamase inhibitor combinations. This study highlights the importance of monitoring the emergence and spread of strains with OmpK36 L3 insertions for the control of resistant KP infections and provides crucial data for drug development and treatment strategies.

microbiology↗

bioRxiv2022

Recurrent emergence of carbapenem resistance in Klebsiella pneumoniae mediated by an inhibitory ompK36 mRNA secondary structure

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

microbiology↗

bioRxiv2019

Genomic analysis of carbapenemase-encoding plasmids from Klebsiella pneumoniae across Europe highlights three major patterns of dissemination

The incidence of Klebsiella pneumoniae infections that are resistant to carbapenems, a last-line class of antibiotics, has been rapidly increasing. The primary mechanism of carbapenem resistance is production of carbapenemase enzymes, which are most frequently encoded on plasmids by blaOXA-48-like, blaVIM, blaNDM and blaKPC genes. Using short-read sequence data, we previously analysed genomes of 1717 isolates from the K. pneumoniae species complex submitted during the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE). Here, we investigated the diversity, prevalence and transmission dynamics of carbapenemase-encoding plasmids using long-read sequencing of representative isolates (n=79) from this collection in combination with short-read data from all isolates. We highlight three major patterns by which carbapenemase genes have disseminated via plasmids. First, blaOXA-48-like genes have spread across diverse lineages primarily via a highly conserved, epidemic pOXA-48-like plasmid. Second, blaVIM and blaNDM genes have spread via transient associations of diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one clonal lineage (ST258/512) despite frequent mobilisation between pre-existing yet diverse plasmids within the lineage. Despite contrasts in these three modes of carbapenemase gene spread, which can be summarised as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage, all are underpinned by significant propagation along high-risk clonal lineages.

microbiology↗