Machine Learning Study in Caries Markers in Oral Microbiota from Monozygotic Twin Children.

In recent years, the etiology of caries has evolved from a simplistic infectious perspective based on Streptococcus mutans and/or Lactobacillus activity, to a multifactorial disease involving a complex oral microbiota, the human genetic background and the environment. The aim of this work was to identify bacterial markers associated with early caries using massive 16S rDNA. To minimize the other factors, the composition of the oral microbiota of twins in which only one of them had caries was compared with their healthy sibling. Twenty-one monozygotic twin pairs without a previous diagnosis of caries were recruited in the context of their orthodontic treatment and divided into two categories: (1) caries group in which only one of the twins had caries; and (2) control group in which neither of the twins had caries. Each participant contributed a single oral lavage sample in which the bacterial composition was determined by 16S rDNA amplification and further high-throughput sequencing. Data analysis included statistical comparison of alpha and beta diversity, as well as differential taxa abundance between groups. Our results show that twins of the control group have a closer bacterial composition than those from the caries group. However, statistical differences were not detected and we were unable to find any particular bacterial marker by 16S rDNA high-throughput sequencing that could be useful for prevention strategies. Although these results should be validated in a larger population, including children from other places or ethnicities, we conclude that the occurrence of caries is not related to the increase of any particular bacterial population.

Epidemiology of the colonization and acquisition of methicillin-resistant staphylococci and vancomycin-resistant enterococci in dogs hospitalized in a clinic veterinary hospital in Spain.

Antibiotic resistance is one of the biggest threats to human and animal health. Methicillin-resistant Staphylococcus spp. (MRS) and vancomycin-resistant Enterococcus spp. (VRE) are of increasing importance in hospital and/or nosocomial infections and represent a potential risk of transmission to humans from infected or colonized companion animals. Studies on the risk factors associated with colonization by multiresistant bacteria in animals are scarce. The present study aimed to estimate the prevalence and incidence of MRS and VRE in canine patients hospitalized in a veterinary hospital and to identify the risk factors for its acquisition and persistence. Nasal and perianal swabs were obtained from 72 dogs. Antimicrobial susceptibility assays and molecular detection of mecA and van genes were performed. A prevalence of 13.9% and incidence of 26.5% was observed in dogs colonized by MRS at hospital admission and release, respectively, higher values than those described in most veterinary studies. Thirty-five Staphylococcus isolates had mecA gene and showed higher resistance levels to most of the antimicrobials evaluated. Previous and concomitant use of antibiotics and corticosteroids has been associated with an increase in MRS colonization. The use of antibiotics in other animals living with the canine patients has also been identified as an associated factor, suggesting cross transmission. The presence of van-resistant genes from Enterococcus spp. was not detected. Pets should be considered possible vehicles of transmission and reservoirs for MRS bacteria and veterinary hospitals should be considered high-risk environments for the occurrence and spread of nosocomial infections and resistant bacteria.

Microcins in Enterobacteriaceae: Peptide Antimicrobials in the Eco-Active Intestinal Chemosphere.

Microcins are low-molecular-weight, ribosomally produced, highly stable, bacterial-inhibitory molecules involved in competitive, and amensalistic interactions between Enterobacteriaceae in the intestine. These interactions take place in a highly complex chemical landscape, the intestinal eco-active chemosphere, composed of chemical substances that positively or negatively influence bacterial growth, including those originated from nutrient uptake, and those produced by the action of the human or animal host and the intestinal microbiome. The contribution of bacteria results from their effect on the host generated molecules, on food and digested food, and organic substances from microbial origin, including from bacterial degradation. Here, we comprehensively review the main chemical substances present in the human intestinal chemosphere, particularly of those having inhibitory effects on microorganisms. With this background, and focusing on Enterobacteriaceae, the most relevant human pathogens from the intestinal microbiota, the microcin's history and classification, mechanisms of action, and mechanisms involved in microcin's immunity (in microcin producers) and resistance (non-producers) are reviewed. Products from the chemosphere likely modulate the ecological effects of microcin activity. Several cross-resistance mechanisms are shared by microcins, colicins, bacteriophages, and some conventional antibiotics, which are expected to produce cross-effects. Double-microcin-producing strains (such as microcins MccM and MccH47) have been successfully used for decades in the control of pathogenic gut organisms. Microcins are associated with successful gut colonization, facilitating translocation and invasion, leading to bacteremia, and urinary tract infections. In fact, Escherichia coli strains from the more invasive phylogroups (e.g., B2) are frequently microcinogenic. A publicly accessible APD3 database http://aps.unmc.edu/AP/ shows particular genes encoding microcins in 34.1% of E. coli strains (mostly MccV, MccM, MccH47, and MccI47), and much less in Shigella and Salmonella (<2%). Some 4.65% of Klebsiella pneumoniae are microcinogenic (mostly with MccE492), and even less in Enterobacter or Citrobacter (mostly MccS). The high frequency and variety of microcins in some Enterobacteriaceae indicate key ecological functions, a notion supported by their dominance in the intestinal microbiota of biosynthetic gene clusters involved in the synthesis of post-translationally modified peptide microcins.

Recombination blurs phylogenetic groups routine assignment in Escherichia coli: setting the record straight.

The characterization of population structures plays a main role for understanding outbreaks and the dynamics of bacterial spreading. In Escherichia coli, the widely used combination of multiplex-PCR scheme together with goeBURST has some limitations. The purpose of this study is to show that the combination of different phylogenetic approaches based on concatenated sequences of MLST genes results in a more precise assignment of E. coli phylogenetic groups, complete understanding of population structure and reconstruction of ancestral clones. A collection of 80 Escherichia coli strains of different origins was analyzed following the Clermont and Doumith's multiplex-PCR schemes. Doumith's multiplex-PCR showed only 1.7% of misassignment, whereas Clermont's-2000 protocol reached 14.0%, although the discrepancies reached 30% and 38.7% respectively when recombinant C, F and E phylogroups were considered. Therefore, correct phylogroup attribution is highly variable and depends on the clonal composition of the sample. As far as population structure of these E. coli strains, including 48 E. coli genomes from GenBank, goeBURST provides a quite dispersed population structure; whereas NeighborNet approach reveals a complex population structure. MLST-based eBURST can infer different founder genotypes, for instance ST23/ST88 could be detected as the founder genotypes for STC23; however, phylogenetic reconstructions might suggest ST410 as the ancestor clone and several evolutionary trajectories with different founders. To improve our routine understanding of E. coli molecular epidemiology, we propose a strategy based on three successive steps; first, to discriminate three main groups A/B1/C, D/F/E and B2 following Doumith's protocol; second, visualization of population structure based on MLST genes according to goeBURST, using NeighborNet to establish more complex relationships among STs; and third, to perform, a cost-free characterization of evolutionary trajectories in variants emerging along the clonal expansion using parsimony methods of phylogenetic analysis.

Normal mutation rate variants arise in a Mutator (Mut S) Escherichia coli population.

The rate at which mutations are generated is central to the pace of evolution. Although this rate is remarkably similar amongst all cellular organisms, bacterial strains with mutation rates 100 fold greater than the modal rates of their species are commonly isolated from natural sources and emerge in experimental populations. Theoretical studies postulate and empirical studies teort the hypotheses that these "mutator" strains evolved in response to selection for elevated rates of generation of inherited variation that enable bacteria to adapt to novel and/or rapidly changing environments. Less clear are the conditions under which selection will favor reductions in mutation rates. Declines in rates of mutation for established populations of mutator bacteria are not anticipated if such changes are attributed to the costs of augmented rates of generation of deleterious mutations. Here we report experimental evidence of evolution towards reduced mutation rates in a clinical isolate of Escherichia coli with an hyper-mutable phenotype due a deletion in a mismatch repair gene, (ΔmutS). The emergence in a ΔmutS background of variants with mutation rates approaching those of the normal rates of strains carrying wild-type MutS was associated with increase in fitness with respect to ancestral strain. We postulate that such an increase in fitness could be attributed to the emergence of mechanisms driving a permanent "aerobic style of life", the negative consequence of this behavior being regulated by the evolution of mechanisms protecting the cell against increased endogenous oxidative radicals involved in DNA damage, and thus reducing mutation rate. Gene expression assays and full sequencing of evolved mutator and normo-mutable variants supports the hypothesis. In conclusion, we postulate that the observed reductions in mutation rate are coincidental to, rather than, the selective force responsible for this evolution.

Polymorphic mutation frequencies of clinical and environmental Stenotrophomonas maltophilia populations.

Mutation frequencies were studied in 174 Stenotrophomonas maltophilia isolates from clinical and nonclinical environments by detecting spontaneous rifampin-resistant mutants in otherwise-susceptible populations. The distribution of mutation frequencies followed a pattern similar to that found for other bacterial species, with a modal value of 1 x 10(-8). Nevertheless, the proportion of isolates showing mutation frequencies below the modal value (hypomutators) was significantly higher for S. maltophilia than those so far reported in other organisms. Low mutation frequencies were particularly frequent among environmental S. maltophilia strains (58.3%), whereas strong mutators were found only among isolates with a clinical origin. These results indicate that clinical environments might select bacterial populations with high mutation frequencies, likely by second-order selection processes. In several of the strong-mutator isolates, functional-complementation assays with a wild-type allele of the mutS gene demonstrated that the mutator phenotype was due to the impairment of MutS activity. In silico analysis of the amino acid changes present in the MutS proteins of these hypermutator strains in comparison with the normomutator isolates suggests that the cause of the defect in MutS might be a H683P amino acid change.

The evolution of contact-dependent inhibition in non-growing populations of Escherichia coli.

In the course of liquid culture, serial passage experiments with Escherichia coli K-12 bearing a mutator gene deletion (DeltamutS) we observed the evolution of strains that appeared to kill or inhibit the growth of the bacteria from where they were derived, their ancestors. We demonstrate that this inhibition occurs after the cells stop growing and requires physical contact between the evolved and ancestral bacteria. Thereby, it is referred to as stationary phase contact-dependent inhibition (SCDI). The evolution of this antagonistic relationship is not anticipated from existing theory and experiments of competition in mass (liquid) culture. Nevertheless, it occurred in the same way (parallel evolution) in the eight independent serial transfer cultures, through different single base substitutions in a gene in the glycogen synthesis pathway, glgC. We demonstrate that the observed mutations in glgC, which codes for ADP-glucose pyrophosphorylase, are responsible for both the ability of the evolved bacteria to inhibit or kill their ancestors and their immunity to that inhibition or killing. We present evidence that without additional evolution, mutator genes, or known mutations in glgC, other strains of E. coli K-12 are also capable of SCDI or sensitive to this inhibition. We interpret this, in part, as support for the generality of SCDI and also as suggesting that the glgC mutations responsible for the SCDI, which evolved in our experiments, may suppress the action of one or more genes responsible for the sensitivity of E. coli to SCDI. Using numerical solutions to a mathematical model and in vitro experiments, we explore the population dynamics of SCDI and postulate the conditions responsible for its evolution in mass culture. We conclude with a brief discussion of the potential ecological significance of SCDI and its possible utility for the development of antimicrobial agents, which unlike existing antibiotics, can kill or inhibit the growth of bacteria that are not growing.

Mutation rate is reduced by increased dosage of mutL gene in Escherichia coli K-12.

A variable but substantial proportion of wild Escherichia coli isolates present consistently lower mutation frequencies than that found in the ensemble of strains. The genetic mechanisms responsible for the hypo-mutation phenotype are much less known than those involved in hyper-mutation. Changes in E. coli mutation frequencies derived from the gene-copy effect of mutS, mutL, mutH, uvrD, mutT, mutY, mutM, mutA, dnaE, dnaQ, and rpoS are explored. When present in a very high copy number ( approximately 300 copies cell(-1)), mutL, mutH, and mutA gene copies yielded >/=twofold decrease in mutation rates determined by Luria-Delbrück fluctuation tests. Nevertheless, when the copy number was not such high ( approximately 15 copies cell(-1)), only mutL results in a consistent twofold decrease in the mutation rate. This reduction seems to be independent from the RecA background, phase of growth, or from the presence of proficient MutS. An increase in mutL gene copies was also able to partially compensate the hypermutator phenotype of a mutS-defective E. coli derivative.

Bacterias con alta tasa de mutación: los riesgos de una vida acelerada / High mutation rate bacteria: Risks of a high-speed life

El proceso evolutivo de un ser vivo se acelera cuanto mayor sea su capacidad para producir variabilidad genética, bien por mutación, bien por recombinación.Sin embargo, cuanto mayor sea esta capacidad, mayor también será el riesgo de acumular mutaciones deletéreas. La variabilidad genética es, por tanto, un proceso altamente regulado, de tal manera que las bacterias tienden a mantener una baja tasa de mutación. En diferentes poblaciones bacterianas analizadas hay siempre un porcentaje variable de cepas con una tasa de mutación superior a la frecuencia modal del resto de la población. Existe una relación directa entre la proporción de cepas que mutan y el grado de estrés del ambiente. Así, en los procesos infecciosos crónicos, en los que el tratamiento antibiótico es constante durante períodos prolongados, se observan los mayores porcentajes de bacterias que mutan, cercano al 50 por ciento de la población. Esta selección positiva de bacterias que mutan es debida al enorme potencial que presentan para desarrollar resistencia antibiótica (100 veces superior a una bacteria normal). Esta capacidad ha sido explotada, en algunos centros de investigación, como un modelo natural de evolución acelerada para predecir la facilidad con la que determinadas variantes resistentes pueden aparecer, saber qué posiciones serán las más susceptibles a los cambios y cuál será el costo para la bacteria. El laboratorio de microbiología debe hacer un esfuerzo por detectar estas cepas mutadoras antes de que desarrollen mecanismos de resistencia e induzcan el fracaso terapéutico

The potential of producing genetic variability, eitherby mutation or by recombination, is the driving for-ce of evolution in a living organism. Geneticvariability is a quite regulated process in which bac-teria tend to maintain a low mutation rate. However,a variable proportion of bacteria with a highermutation rate than that of the modal is alwayspresent in any population. Moreover, a direct rela-tionship exists between the proportion of mutatorstrains and environmental stress. In chronicinfectious diseases, due to prolonged antibioticregimens, nearly 50% of the population may berepresented by mutating bacteria. Such a positiveselection is due to the capacity of this type of strainsto develop antibiotic resistance (100 fold higherthan normal bacteria) This trait has been used asan accelerated-evolution model to predict the easeof certain resistant variants to emerge as well asto infer which targets are more prone to bemodified and the concomitant cost that suchvariability would imply to the organism.The Microbiology laboratory might then doan effort to detect mutating strains before theappearance of resistance mechanisms that maylead to therapeutic failures.Keywords: evolution, genetic recombination,mutagenesis, bacterial drug resistance.Fecha de recepción

Increased mutation frequencies in Escherichia coli isolates harboring extended-spectrum beta-lactamases.

Hypermutable (mutation frequency [f], > or = 4 x 10(-8)) Escherichia coli strains were more frequently found (43%) in a collection of 89 extended-spectrum beta-lactamase (ESBL)-producing isolates from different patients (77 pulsed-field gel electrophoresis clones, 12 ESBL types) than in non-ESBL E. coli (26%) strains (P = 0.03). Among urinary tract isolates, the frequency of hypermutation was 40% in ESBL versus 26% in non-ESBL isolates (P = 0.03).

SOS-independent induction of dinB transcription by beta-lactam-mediated inhibition of cell wall synthesis in Escherichia coli.

Transcription of the dinB gene, encoding DNA polymerase IV, is induced by the inhibition of cell wall synthesis at different levels. Using the beta-lactam antibiotic ceftazidime, a PBP3 inhibitor, as a model, we have shown that this induction is independent of the LexA/RecA regulatory system. Induction of dinB transcription mediated by ceftazidime produces an increase in the reversion of a +1 Lac frameshift mutation.

Fosfomycin and rifampin disk diffusion tests for detection of Escherichia coli mutator strains.

A simple method, using commercial disks to detect Escherichia coli mutator strains, is proposed. The breakpoint for detecting strains with a mutation frequency >/=5 x 10(-7) was established at >/=70 and >/=20 colonies in the inhibition zone of fosfomycin and rifampin disks, respectively, after seeding 100 microl of an overnight culture. Strains with <30 and <10 colonies in fosfomycin and rifampin inhibition zones are presumptively non-mutators.

Polymorphic mutation frequencies in Escherichia coli: emergence of weak mutators in clinical isolates.

Polymorphisms in the rifampin resistance mutation frequency (f) were studied in 696 Escherichia coli strains from Spain, Sweden, and Denmark. Of the 696 strains, 23% were weakly hypermutable (4 x 10(-8) < or = f < 4 x 10(-7)), and 0.7% were strongly hypermutable (f > or = 4 x 10(-7)). Weak mutators were apparently more frequent in southern Europe and in blood isolates (38%) than in urinary tract isolates (25%) and feces of healthy volunteers (11%).

Haemophilus influenzae bla(ROB-1) mutations in hypermutagenic deltaampC Escherichia coli conferring resistance to cefotaxime and beta-lactamase inhibitors and increased susceptibility to cefaclor.

The clinical use of cefaclor has been shown to enrich Haemophilus influenzae populations harboring cefaclor-hydrolyzing ROB-1 beta-lactamase. Such a selective process may lead to the increased use of extended-spectrum cephalosporins or beta-lactams plus beta-lactamase inhibitors and, eventually, resistance to these agents, which has not previously been observed in H. influenzae. In order to establish which bla(ROB-1) mutations, if any, could confer resistance to extended-spectrum cephalosporins and/or to beta-lactamase inhibitors, a plasmid harboring bla(ROB-1) was transformed into hypermutagenic strain Escherichia coli GB20 (DeltaampC mutS::Tn10), and this construct was used in place of H. influenzae bla(ROB-1). Strain GB20 with the cloned gene was submitted to serial passages in tubes containing broth with increasing concentrations of selected beta-lactams (cefotaxime or amoxicillin-clavulanate). Different mutations in the bla(ROB-1) gene were obtained during the passages in the presence of the different concentrations of the selective agents. Mutants resistant to extended-spectrum cephalosporins harbored either the Leu169-->Ser169 or the Arg164-->Trp164 substitution or the double amino acid change Arg164-->Trp164 and Ala237-->Thr237. ROB-1 mutants that were resistant to beta-lactams plus beta-lactamase inhibitors and that harbored the Arg244-->Cys244 or the Ser130-->Gly130 replacement were also obtained. The cefaclor-hydrolyzing efficiencies of the ROB-1 variants were strongly decreased in all mutants, suggesting that if bla(ROB-1) mutants were selected by cefaclor, this drug would prevent the further evolution of this beta-lactamase toward molecular forms able to resist extended-spectrum cephalosporins or beta-lactamase inhibitors.

Frequency of mutation to rifampin resistance in Streptococcus pneumoniae clinical strains: hexA and hexB polymorphisms do not account for hypermutation.

The frequency of mutation to rifampin resistance of 200 clinical Streptococcus pneumoniae isolates was examined. Two peaks were observed in the distribution, with mode frequencies of 2.5 x 10(-7) (20% of isolates) and 2.5 x 10(-8). The hexA and hexB gene entire sequences were analyzed in 13 isolates. Sequences from both hypermutable and "normomutable" strains were conserved relative to that of the R6 S. pneumoniae control strain. The phenotypic Hex system proficiency, in terms of transforming efficiency, was also maintained irrespective of the variations in mutation frequency values.

The regulation of microcin B, C and J operons.

Microcins are ribosomally encoded small peptide antibiotics produced by Gram(-) enterobacteria. Microcin production-biosynthesis, maturation and secretion to the medium-is encoded by gene clusters organized in operons. Production of the best known plasmid-encoded microcins (MccB, MccC and MccJ) switches on when cells reach the stationary growth phase. This production is doubly regulated at transcriptional level by (a). the growth phase: microcin operons silent/repressed during exponential growth are induced/derepressed when cells sense nutrient starvation and stop exponential growth, and (b). global bacterial regulators acting as inducers or repressors of operon expression. The role played by these regulators (CRP, EmrR, IHF, H-NS, LRP, OmpR, Sigma-38 and SpoT) in the expression of specific microcin operons is reviewed.

Very low cefotaxime concentrations select for hypermutable Streptococcus pneumoniae populations.

A mixed culture of an hypermutable hexA Streptococcus pneumoniae mutant strain and its hexA(+) isogenic ancestor was challenged with low cefotaxime concentrations. Despite identical original cefotaxime MICs, the hexA mutant population was significantly selected at very low concentrations, and all of the tested selected variants harbored the Thr550-->Ala mutation in pbp2x. Since cefotaxime selects hypermutators, the risk of secondary acquisition of antibiotic resistance is increased; as expected, the cefotaxime-resistant mutants had a mutation frequency 10 times higher in response to to ciprofloxacin. The present study presents a model (not necessarily reflecting the clinical setting) illustrating the risk of selection of mutators in the evolution of multiple resistance.