Biology subjects

Canton, R.

Publications and source records attributed to Canton, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Spatiotemporal dynamics and stability of multi-kingdom microbial communities in hospital sinks acting as persistent pathogen reservoirs

Hospital sink drains act as persistent reservoirs for nosocomial pathogens, yet the structure and long-term stability of their associated microbial communities remain poorly known. Here, we present an extensive characterization integrating multi-kingdom meta-taxonomic profiling (16S rRNA, ITS, and 18S rRNA, n=93 samples) with culturomics (n>4000 isolates) and phenotypic resistance testing across 14 sinks from a former intensive care unit (ICU). Over a 17-month study period, we tracked community assembly and stability through the renovation and transition from an unpopulated baseline to active clinical use, encompassing both structural remodeling and functional conversion. We showed that former-ICU drains harbor a highly conserved multi-kingdom core that remains resilient despite significant structural changes and periods of inactivity. While a persistent core baseline exists, community composition changes along a continuous temporal gradient driven by structural and anthropogenic shifts. Furthermore, individual sinks assemble as unique microecosystems shaped by local environmental factors. Topological network modeling shows these communities are not random assemblages but integrated ecological units, with clinically relevant, multidrug-resistant WHO-priority pathogens deeply embedded in the network alongside often-overlooked fungi and protozoa. Together, these findings identify hospital sink drains as persistent and structured multi-kingdom microbial reservoirs that harbor clinically relevant multidrug-resistant organisms (MDROs). Their temporal stability and strong sink-specific signatures highlight the need to consider the broader microbial community when monitoring hospital environmental reservoirs and developing infection-control strategies.

microbiology↗

bioRxiv2026

Loss of restriction-modification methyltransferases drives persistence to fluoroquinolones in Pseudomonas aeruginosa

The resurgence of phage therapy has renewed interest in the interplay between phage resistance and antibiotic susceptibility and has stimulated research in the emergent field of non-canonical cellular functions carried out by defence systems. Yet it remains largely unknown whether intracellular antiphage defence systems influence bacterial physiology, resistance or persistence to antibiotics. Here we discovered that besides its canonical antiphage defence function, the type I restriction modification (RM) system profoundly affects the physiology of the opportunistic pathogen Pseudomonas aeruginosa. Deletion of the type I RM methyltransferase HsdM reduces the size of the bacterial nucleoid and delays DNA replication initiation and exit from lag phase in P. aeruginosa PAO1. Crucially, P. aeruginosa strains isolated from patients with cystic fibrosis (CF) and lacking the RM type I system also display slower growth compared to strains isolated from other sites of infections and encoding this system. Deletion of HsdM selectively increases the levels of persisters that survive treatment with fluoroquinolones by displaying enhanced SOS response but without acquiring resistance. Importantly, we measured elevated persistence to fluoroquinolones also in P. aeruginosa CF isolates lacking the type I RM system, providing a functional link between RM systems, slow growth and persistence to fluoroquinolones. Together these findings open a new way of approaching bacterial susceptibility to antibiotics, bringing antiphage defence systems in a forward-facing position in this field.

microbiology↗

bioRxiv2026

Genomic Characterization of the RyC collection: 50 Multidrug Resistant Clinical Isolates of Escherichia coli and Klebsiella spp.

Antimicrobial resistance (AMR) is a major global public health threat, and Enterobacterales producing extended-spectrum {beta}-lactamases (ESBLs) represent some of the most common and concerning pathogens in clinical settings. Importantly, the dissemination of these resistance mechanisms is largely driven by mobile genetic elements (MGEs), particularly plasmids. Advancing our understanding of AMR evolution through experimentation requires moving beyond domesticated laboratory strains and towards clinically relevant isolates. However, despite the abundance of genomic data in public repositories, there is a lack of well-characterised clinical collections available for experimental work. Here, we characterise the RyC collection, which includes 50 multidrug-resistant, ESBL-producing Escherichia coli and Klebsiella spp. strains isolated from the gut microbiota of hospitalised patients at Hospital Universitario Ramon y Cajal (Madrid, Spain). We generated high-quality genome assemblies for all strains using a combination of short- and long-read sequencing technologies. From these data, we performed a comprehensive characterisation of the pangenome, mobilome, resistome and defensome of the collection. We present the RyC collection as a robust and experimentally tractable resource to study AMR evolution and MGEs dynamics in clinically relevant bacterial backgrounds. Impact statementAntimicrobial resistance (AMR) is a growing global health threat driven by the rapid dissemination of resistance genes among clinically relevant bacteria. A major challenge in studying AMR evolution is the reliance on domesticated laboratory strains, which poorly represent the complexity of pathogens circulating in hospitals. Here, we introduce the RyC collection, a set of well-characterised, multidrug-resistant Enterobacterales isolates obtained from hospitalised patients. By combining high-quality genome sequencing with detailed analyses of their gene content and mobile genetic elements (MGEs), this collection provides a realistic and experimentally tractable system to study how resistance evolves and spreads. The RyC collection will facilitate research on AMR dynamics, plasmid biology and host-MGEs interactions, ultimately contributing to the development of more effective strategies to combat antibiotic-resistant infections.

microbiology↗

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

Identification and Functional Insights into New Phage Tail-Like Bacteriocins (PTLBs) Targeting Pseudomonas aeruginosa as new antimicrobials

The current health crisis caused by multidrug resistant (MDR) pathogens is one of the health problems of most concern globally. Infections caused by these pathogens, such as Pseudomonas aeruginosa, lead to high rates of complications, particularly in compromised patients such as cystic fibrosis (CF) patients. The need to counteract and minimize the forecast future impact has led to the rescue of phage therapy. The use of bacteriophages has important advantages, including highly specific targeting, self-amplification at the infection site, minimal disruption of the microbiome, safety and biocompatibility. However, the capacity of bacteria to escape these entities results in a form of resistance that compromises the effectiveness of the therapy. This involves the search for potential alternatives, such as the phage tail-like bacteriocins (PTLBs), also named as tailocins. These high molecular weight particles resemble the tail structure of bacteriophages and are characterized by the absence of genetic material, avoiding the development of resistance, one of the major handicaps associated with phage therapy. In this study, we detected 34 different PTLBs in 75 P. aeruginosa genomes, with different serotypes and sequence types, 11 of which were characterized as novel F-type PTLBs subtypes (F13-F24). Furthermore, we report that four selected PTLBs (R1, F15, F19, R3-F24) can deal with bacterial infection, with the R1 and the F15 PTLBs being the most efficient in clearing infection in vitro, yielding a survival rate of more than 75% in the Galleria mellonella larvae in vivo model. This reaffirms the potential of PTLBs to control P. aeruginosa infections, which can cause chronic infections in some patients, such as people with CF, due to its strong impact as a MDR bacterium. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/671207v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@76b60borg.highwire.dtl.DTLVardef@1a9cd0aorg.highwire.dtl.DTLVardef@d45dd0org.highwire.dtl.DTLVardef@5b345a_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS-The 75 Pseudomonas aeruginosa genomes from people with cystic fibrosis in the study collection included at least one Phage tail-like bacteriocins (PTLB) cluster. -From the 34 different PTLBs detected in the study collection, 7 were R-type, 10 were complex (R and F-type encoded) and 14 were F-type PTLBs. -11 new F-type PTLBs were described in the Pseudomonas aeruginosa collection under study. -An association between the O-antigen present on the surface of the Pseudomonas aeruginosa isolate and the encoded PTLB subtype was detected. -The R1 and F15 PTLBs subtypes display high antimicrobial activity both in vitro and in vivo (Galleria mellonella).

microbiology↗

bioRxiv2025

Competition with Pseudomonas aeruginosa induces Staphylococcus aureus in an antibiotic-tolerant viable but non culturable state

Interactions between different species of pathogenic microbes often affect clinical outcome by altering the virulence or antibiotic resistance of individual microbes. By investigating the interactions between Staphylococcus aureus and Pseudomonas aeruginosa at the population, single-cell and molecular level we discovered that a sub-population of S. aureus enters in a viable non-culturable state that it is not detected via standard microbiology assays. In the presence of P. aeruginosa, S. aureus adopts a survival lifestyle similar to previously described intracellular S. aureus persisters, downregulating nitrogen metabolism and amino acid biosynthesis, while upregulating protein maturation processes. Entrance in a viable but non culturable state is the primary survival strategy of S. aureus in response to vancomycin treatment, whereas only a minority of the S. aureus population survive ciprofloxacin treatment while in a viable but non culturable state. These bacterial interactions may shape the evolution of resistance traits of co-infecting pathogens. Manipulating these interspecies adaptations could provide new opportunities for early therapeutic interventions.

microbiology↗

bioRxiv2024

Anaerobiosis modulates the performance of antimicrobial resistance genes in Enterobacteriaceae

Bacteria must face and adapt to a variety of physicochemical conditions in the environment and during infection. A key condition is the concentration of dissolved oxygen, proportional to the partial pressure of oxygen (PO2), which is extremely variable among environmental biogeographical areas and also compartments of the human and animal body. Here, we sought to understand if the phenotype of resistance determinants commonly found in Enterobacterales can be influenced by oxygen pressure. To do so, we have compared the MIC in aerobic and anaerobic conditions of isogenic Escherichia coli strains containing 136 different resistance genes against 9 antibiotic families. Our results show a complex landscape of changes in the performance of resistance genes in anaerobiosis. Certain changes are especially relevant for their intensity and the importance of the antibiotic family, like the large decreases in resistance observed against ertapenem and fosfomycin among blaVIM {beta}-lactamases and certain fos genes, respectively; however, the blaOXA-48 {beta}-lactamase from the clinically relevant pOXA-48 plasmid conferred 4-fold higher ertapenem resistance in anaerobiosis. Strong changes in resistance patterns in anaerobiosis were also conserved in Klebsiella pneumoniae. Our results suggest that anaerobiosis is a relevant aspect that can affect the action and selective power of antibiotics for specific AMRs in different environments.

microbiology↗

bioRxiv2024

Involvement of Pf-like phages in resistance to phage infection in clinical isolates of P. aeruginosa from cystic fibrosis patients

Pseudomonas aeruginosa is a bacterial pathogen that is a major cause of lung infections in cystic fibrosis (CF) and other patients. Isolates of P. aeruginosa from CF patients commonly carry filamentous phages (Pf phages), a type of temperate phage known to be related to biofilm production and antibiotic sequestration. In this study, 12 new Pf-like phages were identified in a collection of clinical isolates of P. aeruginosa from CF patients. Analysis of the phage genomes revealed different anti-phage defence systems, described here for first time in these types of phages. Finally, relationships between resistance to phage infection and the presence of Pf-like phages and also between each defence system and resistance were observed. IMPORTANCEBacteria harbour a wide range of defence mechanisms to avoid phage infections. These mechanisms hamper the application of phage therapy because they can lead to the rapid acquisition of phage resistance. Temperate phages, including the filamentous phages, carry genes encoding virulence factors and also anti-phage defence mechanisms, as their survival depends on the host survival. In this study, we identified 12 new Pf-like phages encoding several different anti-phage defence mechanisms, some observed for the first time in this type of phage. A relationship between these phages and resistance to phage infection was also observed. The study findings are important as they provide information about newly discovered filamentous phages and their proteomes and also about the role of these phages in resistance to phage infections. Studying the genome of clinical isolates carrying these phages could help to improve phage therapy by targeting these phages or its genes.

microbiology↗

bioRxiv2024

Plasmid-encoded insertion sequences promote rapid adaptation in clinical enterobacteria

Plasmids are extrachromosomal genetic elements commonly found in bacteria. Plasmids are known to fuel bacterial evolution through horizontal gene transfer (HGT), but recent analyses indicate that they can also promote intragenomic adaptations. However, the role of plasmids as catalysts of bacterial evolution beyond HGT remains poorly explored. In this study, we investigate the impact of a widespread conjugative plasmid, pOXA-48, on the evolution of various multidrug-resistant clinical enterobacteria. Combining experimental and within-patient evolution analyses, we unveil that plasmid pOXA-48 promotes bacterial evolution through the transposition of plasmid-encoded insertion sequence 1 (IS1) elements. Specifically, IS1-mediated gene inactivations expedite the adaptation rate of clinical strains in vitro and foster within-patient adaptation in the gut microbiota. We decipher the mechanism underlying the plasmid-mediated surge in IS1 transposition, revealing a negative feedback loop regulated by the genomic copy number of IS1. Given the overrepresentation of IS elements in bacterial plasmids, our findings propose that plasmid-mediated IS transposition represents a crucial mechanism for swift bacterial adaptation.

microbiology↗

bioRxiv2023

β-lactamase expression induces collateral sensitivity in Escherichia coli

Major antibiotic groups are losing effectiveness due to the uncontrollable spread of antimicrobial resistance (AMR) genes. Among these, {beta}-lactam resistance genes -encoding {beta}-lactamases- stand as the most common resistance mechanism in Enterobacterales due to their frequent association with mobile genetic elements. In this context, novel approaches that counter mobile AMR are urgently needed. Collateral sensitivity (CS) occurs when the acquisition of resistance to one antibiotic increases susceptibility to another antibiotic and can be exploited to selectively eliminate AMR. However, most CS networks described so far emerge as a consequence of chromosomal mutations and cannot be leveraged to tackle mobile AMR. Here, we dissected the CS response elicited by the acquisition of a prevalent antibiotic resistance plasmid to reveal that the expression of the {beta}-lactamase blaOXA-48 induces CS to colistin and azithromycin. We next showed that expression of other clinically relevant mobile {beta}-lactamases produces similar CS responses in multiple, phylogenetically unrelated E. coli strains. Finally, by combining experiments with surveillance data comprising thousands of antibiotic susceptibility tests, we showed that {beta}-lactamase-induced CS is pervasive within Enterobacterales. These results highlight that the physiological side-effects of {beta}-lactamases can be leveraged therapeutically, paving the way for the rational design of specific therapies to block mobile AMR or at least counteract their effects.

microbiology↗

bioRxiv2023

Long-term dynamics of the 'Serratia marcescens complex' in the hospital-built environment

Serratia marcescens is an opportunistic pathogen historically associated with abrupt, unpredictable, and severe outbreaks in hospital intensive care units (ICUs) and, more recently, with the spread of acquired genes encoding carbapenem resistance. However, the population biology and ecology of S. marcescens in the hospital ecosystem is still poorly understood. Here, we combine epidemiological information of 1417 Serratia isolates collected from the sinks of a large ICU ward that underwent significant demographic and operational changes (2019-2020) and 99 non-redundant outbreak/non-outbreak isolates from the same hospital (2003-2019), with genomic data of 165 isolates. We first report hospital sinks as reservoirs of heterogeneous and coexistent populations of the S. marcescens complex (SMC). A novel SMC clade congruent with the recently described Serratia nevei species is predominant, exhibiting chromosomal AmpC {beta}-lactamase with an unusual basal expression in contrast to one of the major features of S. marcescens. Persistent Serratia sink strains are identical to those involved in clonal and polyclonal outbreaks of VIM-1 and OXA-48 producers since at least 2017. The "source-sink" dynamics of SMC populations able to acquire the highly conserved plasmids such as IncL carrying blaVIM-1 or blaOXA-48 offer novel insights that might improve interventions to control outbreaks and treat Serratia infections in high-risk hospital areas.

microbiology↗

bioRxiv2023

International and regional spread of carbapenem-resistant Klebsiella pneumoniae in Europe

Klebsiella pneumoniae, an opportunistic pathogen able to cause hospital- and community-acquired infections, is of particular concern due to the active spread of antibiotic resistance genes associated with mobile genetic elements such as plasmids and transposons. Carbapenem-resistant K. pneumoniae (CRKP) exhibit a higher mortality rate than carbapenem-susceptible K. pneumoniae infections. In this COMBACTE-EURECA study, we present genomic data of 687 carbapenem-resistant strains recovered among clinical samples from 41 hospitals in nine Southern European countries (2016-2018) and compared these with the previous EuSCAPE collection (2013-2014). All isolates were resistant (EUCAST criteria) to at least one carbapenem antibiotic. We identified 11 clonal complexes (CCs), with most isolates belonging to the high-risk clones CC258, CC101, CC11, and CC307. blaKPC-like was the most prevalent carbapenemase-encoding gene (45%), along with the dominance of the CC258 lineage. Equally, blaOXA-48 had a wide-ranging spread (39%), and blaNDM was present in half of the ST11 isolates representing a particular lineage circulating in Greece. Two carbapenemase genes were found in 38 isolates (5.5%). Through the combination of both EURECA and EuSCAPE collections, we elucidated the evolution of K. pneumoniae high-risk clones circulating in Europe. Dominating clones and their associated carbapenemase genes exhibit relevant regional differences, namely CC258 in Greece, Italy, and Spain, CC101 present in Serbia and Romania and CC14 in Turkiye. Due to the wide expansion of CRKP, genomic surveillance across Europe with projects such as EURECA provides crucial insights for risk mapping at geo-temporal scales and informs necessary adaptions to the local settings for implementation of control strategies.

microbiology↗

bioRxiv2023

Differences in vertical and horizontal transmission dynamics shape plasmid distribution in clinical enterobacteria

Conjugative plasmids play a key role in the dissemination of antimicrobial resistance (AMR) genes across bacterial pathogens. AMR plasmids are widespread in clinical settings, but their distribution is not random, and certain associations between plasmids and bacterial clones are particularly successful. For example, the globally spread carbapenem resistance plasmid pOXA-48 can use a wide range of enterobacterial species as hosts, but it is usually associated with a small number of specific Klebsiella pneumoniae clones. These successful associations represent an important threat for hospitalized patients. However, knowledge remains limited about the factors determining AMR plasmid distribution in clinically relevant bacteria. Here, we combined in vitro and in vivo experimental approaches to analyze pOXA-48-associated AMR levels and conjugation dynamics in a collection of wild type enterobacterial strains isolated from hospitalized patients. Our results reveal significant variability in these traits across different bacterial hosts, with Klebsiella spp. strains showing higher pOXA-48-mediated AMR and conjugation frequencies than Escherichia coli strains. Using experimentally determined parameters, we developed a simple mathematical model to interrogate the contribution of AMR levels and conjugation permissiveness to plasmid distribution in bacterial communities. The simulations revealed that a small subset of clones, combining high AMR levels and conjugation permissiveness, play a critical role in stabilizing the plasmid in different polyclonal microbial communities. These results help to explain the preferential association of plasmid pOXA-48 with K. pneumoniae clones in clinical settings. More generally, our study reveals that species- and strain-specific variability in plasmid-associated phenotypes shape AMR evolution in clinically relevant bacterial communities. Significance statementConjugative plasmids disseminate AMR genes across bacterial pathogens. Understanding the rules governing plasmid dynamics in bacterial communities is therefore crucial to controlling the global AMR crisis. In this study, we analyzed the dynamics of an AMR plasmid of great clinical relevance, pOXA-48, in a collection of wild type bacteria recovered from hospitalized patients. We reported a high degree of variability in two key plasmid-associated phenotypes, AMR level and conjugation ability, across the collection of clinical bacteria. Using simulations based on the experimental results, we studied how successful associations between AMR plasmids and clinical strains can arise in bacterial communities. Our results revealed that accounting for variability in plasmid-associated phenotypes help to understand the evolution of AMR in clinical settings.

microbiology↗

bioRxiv2022

The Impact of Colistin Resistance on the Activation of Innate Immunity by Lipopolysaccharide Modification

Colistin resistance is caused by different lipopolysaccharide (LPS) modifications, and we propose to evaluate the effect on the innate immune response of in vivo and in vitro colistin resistance acquisition. We used 2 pairs of isogenic strains: (1) Escherichia coli ATCC25922, susceptible to colistin and its isogenic transconjugant-carrying mcr-1 gene; and (2) OXA-48, CTX-M-15 K. pneumoniae susceptible to colistin (CS-Kp) isolated from a urinary infection and its colistin-resistant variant (CR-Kp) from the same patient after prolonged treatment with colistin. No mutation of described genes for colistin resistance (pmrA, pmrB, mgrB. phoP/Q and crrAB) were found in the CR-Kp genome; however, LPS modifications were characterized by negative-ion MALDI-TOF. The strains were co-cultured with human monocytes to determine their survival after phagocytosis and induction to apoptosis. Also, monocytes were stimulated with bacterial LPS to study cytokine and immunecheckpoint production. The addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A of CR-Kp accounted for the colistin resistance. CR-Kp survived significantly longer inside human monocytes after being phagocytosed compared with the CS-Kp strain, whereas no significant differences were observed for the E. coli isogenic strains. In addition, LPS from CR-Kp induced both higher apoptosis in monocytes and higher levels of cytokine and immune checkpoint production than LPS from CS-Kp. This effect was strictly the opposite for E. coli. Our data reveal a variable impact of colistin resistance on the innate immune system, depending on the responsible mechanism. Adding Ara4N to LPS increases bacterial survival after phagocytosis and elicits a higher inflammatory response than its colistin-susceptible counterpart.

microbiology↗

bioRxiv2022

Application of RT-LAMP-CRISPR-Cas13a technology to the detection of OXA-48 producing Klebsiella pneumoniae

Carbapenem-resistant pathogens have been recognized as a health concern because of their ability to cause severe infections and because they are difficult to detect in laboratories. Researchers are making great efforts to develop a diagnostic technique with high levels of sensitivity and specificity, as accurate, early diagnosis is required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic techniques due to their high specificity: Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. This technology is usually combined with isothermal pre-amplification reactions in order to increase the sensitivity of diagnosis. We have developed an RT-LAMP-CRISPR-Cas13a-based assay for the detection of Klebsiella pneumoniae OXA-48 producer strains in clinical samples without the need for RNA extraction. The assay exhibited 100 % specificity, sensitivity, positive predictive value and negative predictive value.

microbiology↗

bioRxiv2022

Within-patient evolution of plasmid-mediated antimicrobial resistance

Antibiotic resistance (AMR) in bacteria is a major threat to public health, and one of the key elements in the spread and evolution of AMR in clinical pathogens is the transfer of conjugative plasmids. The drivers of AMR evolution have been extensively studied in vitro, but the evolution of plasmid-mediated AMR in vivo remains poorly explored. Here, we tracked the evolution of the clinically-relevant plasmid pOXA-48, which confers resistance to the last-resort antibiotics carbapenems, in a large collection of enterobacterial clones isolated from the gut of hospitalised patients. Combining genomic and experimental approaches, we first characterized plasmid diversity and the genotypic and phenotypic effects of multiple plasmid mutations on a common genetic background. Second, using cutting-edge genomic editing in wild-type multidrug resistant enterobacteria, we dissected three cases of within-patient plasmid-mediated AMR evolution. Our results revealed, for the first time, compensatory evolution of plasmid-associated fitness cost, as well as the evolution of enhanced plasmid-mediated AMR, in bacteria evolving within the gut of hospitalised patients. Crucially, we observed that the evolution of plasmid-mediated AMR in vivo involves a pivotal trade-off between resistance levels and bacterial fitness. This study highlights the need to develop new evolution-informed approaches to tackle plasmid-mediated AMR dissemination.

evolutionary biology↗

bioRxiv2021

Discrimination of species within the Enterobacter cloacae complex using MALDI-TOF Mass Spectrometry and Fourier-Transform Infrared Spectroscopy coupled with Machine Learning tools

The Enterobacter cloacae complex (ECC) encompasses heterogeneous clusters of species that have been associated with nosocomial outbreaks. These species may host different acquired antimicrobial resistance and virulence mechanisms and their identification are challenging. This study aims to develop predictive models based on MALDI-TOF MS spectral profiles and machine learning for species-level identification. A total of 198 ECC and 116 K. aerogenes clinical isolates from the University Hospital Ramon y Cajal (Spain) and the University Hospital Basel (Switzerland) were included. The capability of the proposed method to differentiate the most common ECC species (E. asburiae, E. kobei, E. hormaechei, E. roggenkampii, E. ludwigii, E. bugandensis) and K. aerogenes was demonstrated by applying unsupervised hierarchical clustering with PCA pre-processing. We observed a distinctive clustering of E. hormaechei and K. aerogenes and a clear trend for the rest of the ECC species to be differentiated over the development dataset. Thus, we developed supervised, non-linear predictive models (Support Vector Machine with Radial Basis Function and Random Forest). The external validation of these models with protein spectra from the two participating hospitals yielded 100% correct species-level assignment for E. asburiae, E. kobei, and E. roggenkampii and between 91.2% and 98.0% for the remaining ECC species. Similar results were obtained with the MSI database developed recently (https://msi.happy-dev.fr/) except in the case of E. hormaechei, which was more accurately identified by Random Forest. In short, MALDI-TOF MS combined with machine learning demonstrated to be a rapid and accurate method for the differentiation of ECC species.

microbiology↗

bioRxiv2021

Development and Validation of a MALDI-TOF-Based Model to Predict Extended-Spectrum Beta-Lactamase and/or Carbapenemase-Producing in Klebsiella pneumoniae Clinical Isolates

Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) Mass Spectrometry (MS) is a reference method for microbial identification and it can be used to predict Antibiotic Resistance (AR) when combined with artificial intelligence methods. However, current solutions need time-costly preprocessing steps, are difficult to reproduce due to hyperparameter tuning, are hardly interpretable, and do not pay attention to epidemiological differences inherent to data coming from different centres, which can be critical. We propose using a multi-view heterogeneous Bayesian model (KSSHIBA) for the prediction of AR using MALDI-TOF MS data together with their epidemiological differences. KSSHIBA is the first model that removes the ad-hoc preprocessing steps that work with raw MALDI-TOF data. In addition, due to its Bayesian probabilistic nature, it does not require hyperparameter tuning, provides interpretable results, and allows exploiting local epidemiological differences between data sources. To test the proposal, we used data from 402 Klebsiella pneumoniae isolates coming from two different domains and 20 different hospitals located in Spain and Portugal. KSSHIBA outperforms current state-of-the-art approaches in antibiotic susceptibility prediction, obtaining a 0.78 AUC score in Wild Type classification and a 0.90 AUC score in Extended-Spectrum Beta-Lactamases (ESBL)+Carbapenemases (CP)-producers. The proposal consistently removes the need for ad-hoc preprocessing by working with raw MALDI-TOF data, which, in turn, reduces the time needed to obtain the results of the resistance mechanism in microbiological laboratories. The proposed model implementation as well as both data domains are publicly available.

microbiology↗