Biology subjects

Baquero, F.

Publications and source records attributed to Baquero, F..

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

Bacterial Mercury Resistance Reveals a Robust Species-Structured Human Antimicrobial Mobilome

Antimicrobial resistance (AMR) is increasingly recognized as an ecological and evolutionary phenomenon that extends beyond clinical environments. Despite the predominant focus on antibiotics, bacterial metal resistance genes are among the oldest and most widespread adaptive antimicrobial systems, yet their distribution within human-associated microbial communities remains poorly characterized. Here, we investigated the ecology of mercury (Hg) resistance in children from a birth cohort with relatively high Hg exposure. Fecal samples from 234 children aged 4-8 years were analyzed using culture-based screening, whole-genome sequencing, and comparative analyses of metal- and antibiotic-resistance determinants and associated mobile genetic elements (MGEs). Hg-resistance was detected in 79.7% of samples, with HgR Enterobacterales isolated from 57% of children. Hair mercury concentrations were not associated with carriage. Sequencing revealed a phylogenetically diverse collection dominated by Escherichia spp. (61%). Hg resistance was mediated by 79 mer operons primarily associated with Tn21, Tn1696, and Tn5053 families circulating on chromosomes and a highly diverse plasmidome. Both rare and globally distributed plasmids related to foodborne, animal, and clinical Enterobacterales were identified. Metal resistance determinants exhibited strong taxonomic structuring, with Escherichia enriched in iron-uptake systems and siderophores whereas non-Escherichia taxa carried multimetal resistance operons. These findings indicate that Hg-resistance is shaped by ecological interactions and MGEs, becoming partially decoupled from contemporary Hg-exposure and bacterial community composition. The human gut therefore serves as an important reservoir linking environmental metal resistance to the broader evolution of AMR and provides insight into the baseline resistome and plasmidome of human populations.

microbiology↗

bioRxiv2026

The role of the innate immune system in shaping the dynamics of antimicrobial treatment

Contemporary antibiotic treatment almost solely considers the Minimum Inhibitory Concentration (MIC) when deciding to employ an antibiotic, often overlooking the critical role of host immunity. Using Galleria mellonella and a virulent strain of Staphylococcus aureus, we investigate the infection dynamics and treatment outcomes of antibiotics of different classes--including antibiotics to which the bacteria are resistant--and a lytic bacteriophage. Surprisingly, we find that the hosts ability to control bacterial density and survive infection does not depend on the specific type of antimicrobial agent nor the bacterias susceptibility to it. Our results demonstrate that the innate immune system is the primary factor in therapeutic success, capable of clearing even highly resistant infections, such as those involving beta-lactamase-producing or ribosomal mutant-resistant strains. These findings challenge the traditional antibiotic-centric view of infection outcomes and emphasize the need to account for host-pathogen-drug interactions beyond simple MIC measurements when designing clinical treatment regimens.

microbiology↗

bioRxiv2025

The Antimicrobial Gut Resistome of the Wayampi reveals a shared background of antibiotic and metal resistance genes with industrialised populations, underscoring the robust-yet-fragile architecture of human gut microbiomes.

BackgroundMetagenomics enables detailed profiling of antimicrobial resistance genes, but most studies focus solely on antibiotic resistance genes (ARGs), use low-sensitivity methods, and rarely investigate populations with minimal anthropogenic pollution. We analyzed fecal resistomes of 95 Wayampi individuals, an Indigenous community in remote French Guiana, using a targeted metagenomic capture platform covering 8,667 resistance genes to antibiotics (ARGs), metals (MRGs), and biocides (BRGs) (PMID: 29335005). Resistome profiles were compared with Europeans to assess population-level differences. ResultsARG richness was similar between groups (259 in Wayampi vs. 264 in Europeans, 159 shared), but MRGs + BRGs (BAC) gene richness was signifcantly higher in Wayampi (3,670 vs. 2,039). Most genes appeared in a minority of individuals (mean 5% for ARGs, 2% for BAC), but several ARGs for tetracyclines [tet(32), tet(40), tet(O), tet(Q), tet(W), tet(X), tetAB(P)], aminoglycosides (ant6'-I, aph3-III), macrolides (ermB, ermF, mefA), and sulfonamides (sul2) were present in all individuals. Tetracycline resistance genes predominated overall, while beta-lactam resistance genes were more common in Wayampi, and genes conferring resistance to aminoglycosides, amphenicols, and folate inhibitors were more frequent in Europeans. Among MRGs, copper and arsenic resistance genes prevailed, followed by those for zinc, iron, cobalt, and, nickel. Up to 76% of Wayampiis carried acquired MRGs for copper (pcoABCDRS and tcrB), silver (silACFPRS), arsenic (ars), and mercury (mer) detoxification. Shannon diversity indices were similar for ARGs, MRGs, and BRGs, but composition and evenness differed significantly. UMAP and ADONIS analyses distinguised cohorts by ARG profiles (p < 0.001), but not MRGs or BRGs. Correlation analysis revealed conserved gene-sharing networks and introgression of acquired ARGs and MRGs in both gut microbiomes. ConclusionsThe diverse and balanced Wayampi resistome reflect a less perturbed microbiome compared to industrialized populations, and reveals a background of core and shell acquired ARGs and MRGs, consistent with the "robust-yet-fragile" architecture of scale-free networks. The patchy yet resilient distribution of resistance genes reflects varying levels of conserved gene sharing highways probably shaped by long-term microbial-human evolution, and support a broader view of acquired antimicrobial resistance.

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

The Tradeoffs Between Persistence and Mutation Rates at Sub-Inhibitory Antibiotic Concentrations in Staphylococcus aureus

The rational design of the antibiotic treatment of bacterial infections employs these drugs to reach concentrations that exceed the minimum needed to prevent the replication of the target bacteria. However, within a treated patient, spatial and physiological heterogeneity promotes antibiotic gradients such that the concentration of antibiotics at specific sites is below the minimum needed to inhibit bacterial growth. Here, we investigate the effects of sub-inhibitory antibiotic concentrations on three parameters central to bacterial infection and the success of antibiotic treatment, using in vitro experiments with Staphylococcus aureus and mathematical-computer simulation models. Our results, using drugs of six different classes, demonstrate that exposure to sub-inhibitory antibiotic concentrations not only alters the dynamics of bacterial growth but also increases the mutation rate to antibiotic resistance and decreases the rate of production of persister cells thereby reducing the persistence level. Understanding this trade-off between mutation rates and persistence levels resulting from sub-inhibitory antibiotic exposure is crucial for optimizing, and mitigating the failure of, antibiotic therapy.

microbiology↗

bioRxiv2023

Bdellovibrio's Prey-Independent Growth is Fuelled by Amino Acids as a Carbon Source

Identifying the nutritional requirements and growth conditions of microorganisms is crucial for determining their applicability in industry and understanding their role in clinical ecology. Predatory bacteria such as Bdellovibrio bacteriovorus have emerged as promising tools for combating infections by human bacterial pathogens due to their natural killing features. Bdellovibrios lifecycle occurs inside prey cells, using the cytoplasm as a source of nutrients and energy. However, this lifecycle supposes a challenge when determining the specific uptake of metabolites from the prey to complete the growth inside cells, a process that has not been completely elucidated. Here, following a model-based approach we illuminate the ability of Bdellovibrio bacteriovorus to replicate DNA, increase biomass, and generate adenosine triphosphate (ATP) in an amino acid-based rich media in the absence of prey, keeping intact its predatory capacity. In this culture, we determined the main carbon sources used and their preference, being glutamate, serine, aspartate, isoleucine, and threonine. This study offers new insights into the role of predatory bacteria in natural environments and establishes the basis for developing new Bdellovibrio applications using appropriate metabolic and physiological methodologies.

microbiology↗

bioRxiv2023

The Evolution of Heteroresistance via Small Colony Variants in Escherichia coli Following Long Term Exposure to Bacteriostatic Antibiotics

Traditionally, bacteriostatic antibiotics are agents able to arrest bacterial growth. Despite being traditionally viewed as unable to kill bacterial cells, when they are used clinically the outcome of these drugs is frequently as effective as when a bactericidal drug is used. We explore the dynamics of Escherichia coli after exposure to two ribosome-targeting bacteriostatic antibiotics, chloramphenicol and azithromycin, for thirty days. The results of our experiments provide evidence that bacteria exposed to these drugs replicate, evolve, and generate a sub-population of small colony variants (SCVs) which are resistant to multiple drugs. These SCVs contribute to the evolution of heteroresistance and rapidly revert to a susceptible state once the antibiotic is removed. Stated another way, exposure to bacteriostatic drugs selects for the evolution of heteroresistance in populations previously lacking this trait. More generally, our results question the definition of bacteriostasis as populations exposed to bacteriostatic drugs are replicating despite the lack of net growth.

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

Theoretical Considerations and Empirical Predictions of the Pharmaco- and Population Dynamics of Heteroresistance

Antibiotics are considered one of the most important contributions to clinical medicine in the last 100 years. Due to the use and overuse of these drugs, there have been increasing frequencies of infections with resistant pathogens. One form of resistance, heteroresistance, is particularly problematic; pathogens appear sensitive to a drug by common susceptibility tests. However, upon exposure to the antibiotic, resistance rapidly ascends, and treatment fails. To quantitatively explore the processes contributing to the emergence and ascent of resistance during treatment and the waning of resistance following cessation of treatment, we develop two distinct mathematical and computer-simulations models of heteroresistance. In our analysis of the properties of these models, we consider the factors that determine the response to antibiotic-mediated selection. In one model, heteroresistance is progressive, with each resistant state sequentially generating a higher resistance level. In the other model, heteroresistance is non-progressive, with a susceptible population directly generating populations with different resistance levels. The conditions where resistance will ascend in the progressive model are narrower than those of the non-progressive model. The rates of reversion from the resistant to the sensitive states are critically dependent on the transition rates and the fitness cost of resistance. Our results demonstrate that the standard test used to identify heteroresistance is insufficient. The predictions of our models are consistent with empirical results. Our results demand a reevaluation of the definition and criteria employed to identify heteroresistance. We recommend the definition of heteroresistance should include a consideration of the rate of return to susceptibility. Significance StatementThis mathematical modeling and computer-simulation study quantitatively explores two broadly different, previously undescribed, classes of heteroresistance. In our analysis of the properties of these models, we consider the response of heteroresistant populations to antibiotic exposure, focusing on the conditions where heteroresistance could lead to clinical treatment failure. We also provide novel consideration to the rate of reversion from a resistant to sensitive state. Our analysis illustrates the need to include the reversion rate from resistant to sensitive in the definition of heteroresistance and questions the sufficiency of the method currently used to identify heteroresistance.

microbiology↗

bioRxiv2022

What's the matter with MICs: The contribution of nutrients and limiting resources to the pharmacodynamics of antibiotics and bacteria

The minimum inhibitory concentration (MIC) of an antibiotic required to prevent replication is used both as a measure of the susceptibility/resistance of bacteria to that drug and as the single pharmacodynamic parameter for the rational design of antibiotic treatment regimes. MICs are estimated in vitro under conditions optimal for the action of the antibiotic. However, bacteria rarely grow in these optimal conditions. Using a mathematical model of the pharmacodynamics of antibiotics, we make predictions about the nutrient dependency of bacterial growth in the presence of antibiotics. We test these predictions with experiments in a rich media and a glucose-limited minimal media with Escherichia coli and eight different antibiotics. Our experiments uncover properties that question the sufficiency of using MICs and simple pharmacodynamic functions as measures of the pharmacodynamics of antibiotics under the nutritional conditions of infected tissues. To an extent that varies among drugs: (i) The estimated MICs obtained in rich media are greater than those estimated in minimal media. (ii) Exposure to these drugs increases the time before logarithmic growth starts, their lag. (iii) The stationary phase density of E. coli populations declines with greater sub-MIC antibiotic concentrations. We postulate a mechanism to account for the relationship between the sub-MIC concentration of antibiotics and the stationary phase density of bacteria and provide evidence in support of this hypothesis. We discuss the implications of these results to our understanding of the MIC as the unique pharmacodynamic parameter used to design protocols for antibiotic treatment.

microbiology↗

bioRxiv2021

Predation Efficiency upon Clinical Isolates: Bdellovibrio bacteriovorus is Prey Specific and Origin Dependent

The use of predatory bacteria as live antibiotics has been proposed for managing bacterial infections, especially for those caused by antibiotic multiresistant isolates for which there are few therapeutic options. However, the current knowledge in this field is scarce, with most of the available data based on environmental isolates, with a significant lack of human clinical samples. In this study, we evaluated the predatory spectrum of the reference strain Bdellovibrio bacteriovorus 109J on 13 Serratia marcescens (5 of which were carbapenemase producers) and 78 Pseudomonas aeruginosa clinical isolates from respiratory (colonizing the lungs of patients with cystic fibrosis) or bacteremic infections, differentiated by phenotype (mucoid or not), antibiotic resistance phenotype (including multidrug-resistant isolates), and genetic lineage (frequent and rare sequence types). The source of the isolates was significantly associated with predation efficiency (100% for S. marcescens, 67% for P. aeruginosa from cystic fibrosis, and 25% for P. aeruginosa from bacteremia). In contrast, no correlation with colonial morphotype, genetic background, or antibiotic susceptibility was found. To evaluate the influence of the predator on the predation event, we employed a more aggressive B. bacteriovorus mutant 109J preying upon the same 48 bacteremic P. aeruginosa isolates. The mutants predation efficiency was higher than that of their wild-type counterpart (43% vs. 25%), pointing out that predation is specific to each prey-predator pair of isolates. Our results provide the most extensive study of clinical prey susceptibility published to date and show that the prey-predator interaction is influenced by the origin of the isolates rather than by their genetic background or their antibiotic susceptibility phenotype. IMPORTANCEThe potential usefulness of predatory bacteria in controlling human pathogens, particularly those that are multiresistant to antibiotics, is enormous. Although this possibility has long been suggested, there are still no data on predation susceptibility in clinical strains, and the possible presence of autochthonous predators of the human microbiota has not been investigated. In this study, we employed a reference predator with an environmental origin to study predation phenomena in 3 well-characterized collections of human clinical isolates. Our results demonstrated that predation is a specific consequence of each prey-predator interaction, with the origin of the strains the most relevant factor. In contrast, the genetic background, morphotype, and antibiotic resistance did not appear to influence the predation phenomenon. We also highlight the involvement of a putative polyhydroxyalkanoate depolymerase protein of B. bacteriovorus in determining prey susceptibility. To our knowledge, this study is the largest performed with strains of clinical origin, discriminating between various genera and including strains with multiresistance to antibiotics.

ecology↗

bioRxiv2021

PATO: Pangenome Analysis Toolkit

MotivationComparative genomics is a growing field but one that will be eventually overtaken by sample size studies and the increase of available genomes in public databases. We present the Pangenome Analysis Toolkit (PATO) designed to simultaneously analyze thousands of genomes using a desktop computer. The tool performs common tasks of pangenome analysis such as core-genome definition and accessory genome properties and includes new features that help characterize population structure, annotate pathogenic features and create gene sharedness networks. PATO has been developed in R to integrate with the large set of tools available for genetic, phylogenetic and statistical analysis in this environment. ResultsPATO can perform the most demanding bioinformatic analyses in minutes with an accuracy comparable to state-of-the-art software but 20-30x times faster. PATO also integrates all the necessary functions for the complete analysis of the most common objectives in microbiology studies. Lastly, PATO includes the necessary tools for visualizing the results and can be integrated with other analytical packages available in R. AvailabilityThe source code for PATO is freely available at https://github.com/irycisBioinfo/PATO under the GPLv3 license. Contactval.fernandez@salud.madrid.org Supplementary informationSupplementary data are available at Bioinformatics online

bioinformatics↗

bioRxiv2021

Fitness cost of vancomycin-resistant Enterococcus faecium plasmids associated with hospital infection outbreaks

BackgroundVancomycin resistance is mostly associated with Enterococcus faecium due to Tn1546-vanA located on narrow- and broad-host plasmids of various families. The studys aim was to analyse the effects of acquiring Tn1546-plasmids with proven epidemicity in different bacterial host backgrounds. MethodsWidespread Tn1546-plasmids of different families RepA_N (n=5), Inc18 (n=4) and/or pHT{beta} (n=1), and prototype plasmids RepA_N (pRUM) and Inc18 (pRE25, pIP501) were analysed. Plasmid transferability and fitness cost were assessed using E. faecium (GE1, 64/3) and Enterococcus faecalis (JH2-2/FA202/UV202) recipient strains. Growth curves (Bioscreen C) and Relative Growth Rates were obtained in presence/absence of vancomycin. Plasmid stability was analysed (300 generations). Whole genome sequencing (Illumina-MiSeq) of non-evolved and evolved strains (GE1/64/3 transconjugants, n=49) was performed. SNP calling (breseq software) of non-evolved strains was used for comparison. ResultsAll plasmids were successfully transferred to different E. faecium clonal backgrounds. Most Tn1546-plasmids and Inc18 and RepA_N prototypes reduced host fitness (-2%-18%) while the cost of Tn1546 expression varied according to the Tn1546-variant and the recipient strain (9-49%). Stability of Tn1546-plasmids was documented in all cases, often with loss of phenotypic resistance and/or partial plasmid deletions. Point mutations and/or indels associated with essential bacterial functions were observed on the chromosome of evolved strains, some of them linked to increased fitness. ConclusionsThe stability of E. faecium Tn1546-plasmids in the absence of selective pressure and the high intra-species conjugation rates might explain the persistence of vancomycin resistance in E. faecium populations despite the significant burden they might impose on bacterial host strains.

microbiology↗

bioRxiv2020

A 21-year survey of from bloodstream infections (BSIs) in a tertiary hospital reveals how community-hospital dynamics, influence local BSI rates, the trends of the B2 phylogroup and the STc131 pandemic clone

This is a longitudinal study comprising 649 Escherichia coli (EC) isolates representing all 7165 EC-BSI episodes recorded in a hospital (1996-2016). Strains analysis included clonal identification (phylogenetic groups/subgroups, STc131 subclades, PFGE, and WGS), antibiotic susceptibility (13 antibiotics), and virulence-associated genes (VAGs, 29 genes). The incidence of BSI-EC increased from 1996 to 2016 (5.5 to 10.8 BSI episodes/1000 hospitalizations, average 7-8/1000). B2 isolates predominate (53%), subgroups B2-I (STc131), B2-II, B2-IX, and B2-VI representing 25%, 25%, 14%, and 9%, respectively. Intertwined waves of community-acquired (CA) + healthcare-associated and community-onset healthcare-associated (HCA), and hospital-acquired (HA) episodes of both B2 and non-B2 phylogroups occurred. A remarkable increase was only observed for B2-I-STc131 (C1/C2 subclades), with oscillations for other B2 subgroups and phylogroups throughout the years. Epidemic and persistent clones (comprising isolates with highly similar/identical-PFGE types and genomes differing in 18-97 SNPs) of B2-I (STc131), B2-II (STc73), B2-III (STc127), B2-IX (STc95), and B2-VI (STc12) were recovered from different patients, most at hospital admission, for long periods (2-17 years), ESBL producers or resistance to ciprofloxacin in B2 isolates were almost restricted to B2-I (STc131) subclade C. STc131 contributed to increasing the B2 rates but only transiently altered the EC-population structure. The increase of EC-BSI was determined by waves of CA+HCA-BSI episodes that predate the waves of HA-BSI. Besides the risk of hospital transmission that led to temporal increases in BSIs, this study suggests that EC-populations/clones from community-based healthy individuals may occasionally have an epidemic structure and provide a source of transmissible strains influencing the HA-BSIs incidence. IMPORTANCESepsis is the third cause of mortality in Western countries and one of the Global Health threads recognized by the WHO since 2017. Despite Escherichia coli constitutes the most common cause of bloodstream infections (BSI), its epidemiology is not fully understood, in part due to the scarcity of local and longitudinal studies. Our work analyzes the long-term dynamics of E. coli causing bacteremia in a single institution and reveals waves of different clonal lineages that emerge periodically and successfully spread afterward in both the community and hospitals. Because the origin of BSI-E. coli infections is the gut, the microbiota of healthy individuals might occasionally have an epidemic structure, providing a source of E. coli strains to influence the incidence of hospital BSIs. The study complements previous fractionated observations focusing on specific E. coli lineages or antibiotic-resistant isolates in the last decades and helps to understand the epidemiology of E. coli BSIs and the dynamics of pandemic clones.

microbiology↗

bioRxiv2020

Simulating the Influence of Conjugative Plasmids Kinetic Values on the Multilevel Dynamics of Antimicrobial Resistance in a Membrane Computing Model

Plasmids harboring antibiotic resistance genes differ in their kinetic values as plasmid conjugation rate, segregation rate by incompatibility with related plasmids, rate of stochastic loss during replication, cost reducing the host-cell fitness, and frequency of compensatory mutations to reduce plasmid cost, depending on the cell mutation frequency. How variation in these values influence the success of a plasmid and their resistance genes in complex ecosystems, as the microbiota? Genes are located in plasmids, plasmids in cells, cells in populations. These populations are embedded in ensembles of species in different human hosts, are able to exchange between them bacterial ensembles during cross-infection and are located in the hospital or the community setting, under various levels of antibiotic exposure. Simulations using new membrane computing methods help predict the influence of plasmid kinetic values on such multilevel complex system. In our simulation, conjugation frequency needed to be at least 10-3 to clearly influence the dominance of a strain with a resistant plasmid. Host strains able to stably maintain two copies of similar plasmids harboring different resistances, coexistence of these resistances can occur in the population. Plasmid loss rates of 10-4 or 10-5 or plasmid fitness costs [≥]0.06 favor the plasmids located in the most abundant species. The beneficial effect of compensatory mutations for plasmid fitness cost is proportional to this cost, only at high mutation frequencies (10-3-10-5). Membrane computing helps set a multilevel landscape to study the effect of changes in plasmid kinetic values on the success of resistant organisms in complex ecosystems.

microbiology↗

bioRxiv2018

Reconstruction of Escherichia coli ancient diversification by layered phylogenomics and polymorphism fingerprinting

The rapidly increasing availability of whole genomes provides the opportunity to reach an updated comprehensive view of bacterial evolution. The staggered diversification of evolutionary processes, based on the combined strategy of layered phylogenomics and polymorphism fingerprinting, give a new perspective in phylogenetic reconstructions. Layered phylogenomics is based on the assignation of genes according to five different evolutionary layers: minimal genome, genus-core genome, species-core genome, phylogroup-core genome and phylogroup-flexible genome. Polymorphism fingerprinting is based on the detection of conserved positions in each phylogenetic group but differing from those of their hypothetical ancestors. This approach was applied to Escherichia coli because there are unresolved evolutionary questions, although has been highly studied. Phylogenetic analysis based on 6,220 full genomes, identified three E. coli root lineages, defined as D, EB1A and FGB2. A new phylogroup, called G was detected near to phylogroup B2. The closest phylogroup to ancestral E. coli was phylogroup D, whereas E and F were the closest ones in their respective lineages; moreover, A and B2 were the most distant phylogroups in EB1A and FGB2 respectively. We suspect that EB1A and FGB2 lineages represent different adaptive strategies. In the deepest branch of EB1A lineage, the number of accumulated mutations was lower than in recent branches, whereas in FGB2 lineage the opposite occurred. The FGB2 lineage was enriched in genes related to host colonization-pathogenicity and toxin-antitoxin systems (such as hipA), whereas B1A sub-lineage acquired functions related to uptake and metabolism of carbohydrates (such as bgl, mng or xlyE). This new combined strategy shows a detailed staggered evolutionary reconstruction, which help us to understand the deepest events and the selection forces have driven E. coli diversification. This approach could add resolution in the reconstruction of the evolutionary trajectories of other microorganisms.\n\nAuthor summaryPhylogeny based on whole genome provides the opportunity to study the history of eco-adaptive diversification of any bacterial taxon. Different strategies have been proposed for knowing the evolutionary trajectories in some species, such as Escherichia coli; however, these analyses were based on a limited number of sequences, and sometimes the evolutionary reconstructions reached clashed positions, especially in the ancestral inferences. For adding resolution in evolutionary reconstructions, we propose a combination of approaches, such as layered phylogenomics based on the use of different set of genes corresponding to the successive evolutionary steps, and polymorphism fingerprinting which detects hallmarks of the ancient mutations. We propose to use E. coli because it is paradigmatic example of the evolutionary inconsistences despite being a microorganism with enough evolutionary analysis. Three ancestral lineages were established with this strategy and the staggered reconstruction about the origin and diversification of E. coli phylogroups was inferred. Moreover, in the context of this study, a new E. coli phylogroup was defined. The main lineages represent different adaptive strategies, one lineage gained genes involved in pathogenicity, and another one acquired genes allowing the obtainment of energy from different sources.

microbiology↗

bioRxiv2018

Antibiotic killing of diversely generated populations of non-replicating bacteria

Non-replicating bacteria are known to be (or at least commonly thought to be) refractory to antibiotics to which they are genetically susceptible. Here, we explore the sensitivity to antibiotic-mediated killing of three classes of non-replicating populations of planktonic bacteria (1) stationary phase, when the concentration of resources and/or nutrients are too low to allow for population growth; (2) persisters, minority subpopulations of susceptible bacteria surviving exposure to bactericidal antibiotics; (3) antibiotic-static cells, bacteria exposed to antibiotics that prevent their replication but kill them slowly if at all, the so-called bacteriostatic drugs. Using experimental populations of Staphylococcus aureus Newman and Escherichia coli K12 and respectively 14 and 11 different antibiotics, we estimate the rates at which these drugs kill these different types of non-replicating bacteria. Contrary to the common belief that bacteria that are non-replicating are refractory to antibiotic-mediated killing, all three types of non-replicating populations of these Gram-positive and Gram-negative bacteria are consistently killed by aminoglycosides and the peptide antibiotics, daptomycin and colistin, respectively. This result indicates that non-replicating cells, irrespectively of why they do not replicate, have an almost identical response to the cidal activity of antibiotics. We discuss the implications of these results to our understanding of the mechanisms of action of antibiotics and the possibility of adding a short-course of aminoglycosides or peptide antibiotics to conventional therapy of bacterial infections.

microbiology↗