Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure

One of the main hurdles for the development of an effective and broadly protective vaccine against nonencapsulated isolates of Haemophilus influenzae (NTHi) lies in the genetic diversity of the species, which renders extremely difficult the identification of cross-protective candidate antigens. To assess whether a population structure of NTHi could be defined, we performed genome sequencing of a collection of diverse clinical isolates representative of both carriage and disease and of the diversity of the natural population. Analysis of the distribution of polymorphic sites in the core genome and of the composition of the accessory genome defined distinct evolutionary clades and supported a predominantly clonal evolution of NTHi, with the majority of genetic information transmitted vertically within lineages. A correlation between the population structure and the presence of selected surface-associated proteins and lipooligosaccharide structure, known to contribute to virulence, was found. This high-resolution, genome-based population structure of NTHi provides the foundation to obtain a better understanding, of NTHi adaptation to the host as well as its commensal and virulence behavior, that could facilitate intervention strategies against disease caused by this important human pathogen.The Gram-negative bacterium Haemophilus influenzae colonizes the human nasopharynx and can cause a spectrum of diseases (1). Members of this species can be separated into those that are encapsulated and those that do not express a capsule, so-called nontypeable H. influenzae (NTHi) (2). Encapsulated strains belong to one of six distinct capsular serotypes (a, b, c, d, e, and f) of which type b strains are notoriously associated with invasive disease (3). NTHi are associated with common pediatric diseases, including otitis media (OM) (4, 5), and with exacerbations of chronic obstructive pulmonary disease (COPD) in adults (6).Although capsule-based vaccines against serotype b strains exist, NTHi vaccine candidates containing outer-membrane proteins have been unsuccessful due to their inability to induce functional antibodies to epitopes representative of the phenotypic variation within the population, resulting in poor coverage against heterologous strains (7–9). To devise containment strategies based on vaccination, it is therefore essential to characterize the population structure of the NTHi strains and their genomic variability. Classification schema based on ribotyping (10), multilocus enzyme electrophoresis (11, 12), and multilocus sequence typing (MLST) (13–15) have shown that isolates of encapsulated H. influenzae could be classified into a small number of monophyletic lineages, with reduced diversity (12, 16) and genetically distinct from NTHi strains, that constitute the vast majority of the circulating population (11). Despite these efforts, there is still a substantial lack of knowledge regarding the structure of the NTHi population, mainly attributable to the impact that homologous recombination has on the evolution of the genomes of this pathogen, which is higher in NTHi compared with capsulated strains (15). So far, data on isolates from carriers and those with disease have shown little correlation between MLST typing and the clinical source or the geographical origin of the strains studied (17).Whole-genome sequencing can be used to characterize the population structure of large collections of isolates of bacterial pathogens (18–20) and to study the microevolution of virulent lineages (21, 22). Here, we use whole-genome sequencing of NTHi isolates of diverse clinical and geographical origin to assess population structure. Analysis of single nucleotide polymorphisms (SNPs) revealed six statistically supported clusters of isolates that correlated with the composition of the accessory genome. Our data lay the foundation for a comprehensive definition of the population structure of NTHi that can underpin the development of strategies to fight NTHi-associated disease.     

Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination

Molecular data on a limited number of chromosomal loci have shown that the population of Neisseria meningitidis (Nm), a deadly human pathogen, is structured in distinct lineages. Given that the Nm population undergoes substantial recombination, the mechanisms resulting in the evolution of these lineages, their persistence in time, and the implications for the pathogenicity of the bacterium are not yet completely understood. Based on whole-genome sequencing, we show that Nm is structured in phylogenetic clades. Through acquisition of specific genes and through insertions and rearrangements, each clade has acquired and remodeled specific genomic tracts, with the potential to impact on the commensal and virulence behavior of Nm. Despite this clear evidence of a structured population, we confirm high rates of detectable recombination throughout the whole Nm chromosome. However, gene conversion events were found to be longer within clades than between clades, suggesting a DNA cleavage mechanism associated with the phylogeny of the species. We identify 22 restriction modification systems, probably acquired by horizontal gene transfer from outside of the species/genus, whose distribution in the different strains coincides with the phylogenetic clade structure. We provide evidence that these clade-associated restriction modification systems generate a differential barrier to DNA exchange consistent with the observed population structure. These findings have general implications for the emergence of lineage structure and virulence in recombining bacterial populations, and they could provide an evolutionary framework for the population biology of a number of other bacterial species that show contradictory population structure and dynamics.     

Trial-to-trial variability in the responses of neurons carries information about stimulus location in the rat whisker thalamus

From the perspective of neural coding, the considerable trial-to-trial variability in the responses of neurons to sensory stimuli is puzzling. Trial-to-trial response variability is typically interpreted in terms of "noise" (i.e., it represents either intrinsic noise of the system or information unrelated to the stimuli). However, trial-to-trial response variability can be considerably different across stimuli, suggesting that it could also provide an important contribution to the information conveyed by the neural responses about the stimuli. To test this hypothesis, we addressed the problem of discriminating stimulus location from the spike-count responses of neurons recorded in the ventro-postero-medial (VPM) nucleus of the thalamus in anesthetized rats. Using a recently developed information theory approach, we verified that differences between stimuli in the trial-to-trial spike-count variability of the responses provided an important contribution to the overall information carried by the neurons. In addition, we found that the relatively reliable (sub-Poisson) firing regime of our VPM neurons was not only more informative, but also more redundant between neurons compared with a more variable (Poisson) firing regime with the same total number of spikes. The typical increase in trial-to-trial response variability from the periphery to the cortex could therefore serve as a strategy to reduce redundancy between neurons and promote efficient sparse coding distributed in large populations of neurons. Overall, our data suggest that the trial-to-trial response variability plays a critical role in establishing the trade-off between total information and redundancy between neurons in population codes.     

Functional role of exercise-induced cortical organization of sensorimotor cortex after spinal transection

Spinal cord transection silences neuronal activity in the deafferented cortex to cutaneous stimulation of the body and untreated animals show no improvement in functional outcome (weight-supported stepping) with time after lesion. However, adult rats spinalized since neonates that receive exercise therapy exhibit greater functional recovery and exhibit more cortical reorganization. This suggests that the change in the somatotopic organization of the cortex may be functionally relevant. To address this issue, we chronically implanted arrays of microwire electrodes into the infragranular layers of the hindlimb somatosensory cortex of adult rats neonatally transected at T8/T9 that received exercise training (spinalized rats) and of normal adult rats. Multiple, single neuron activity was recorded during passive sensory stimulation, when the animals were anesthetized, and during active sensorimotor stimulation during treadmill-induced locomotion when the animal was awake and free to move. Our results demonstrate that cortical neurons recorded from the spinalized rats that received exercise 1) had higher spontaneous firing rates, 2) were more likely to respond to both sensory and sensorimotor stimulations of the forelimbs, and also 3) responded with more spikes per stimulus than those recorded from normal rats, suggesting expansion of the forelimb map into the hindlimb map. During treadmill locomotion the activity of neurons recorded from neonatally spinalized rats was greater during weight-supported steps on the treadmill compared with the neuronal activity during nonweight supported steps. We hypothesize that this increased activity is related to the ability of the animal to take weight supported steps and that, therefore, these changes in cortical organization after spinal cord injury are relevant for functional recovery.     

Repeatability,Completion Time,and Predictive Ability of Four Diabetes-Related Foot Ulcer Classification Systems

Introduction:The inter and intra-observer reproducibility of measuring the Wound Ischemia foot Infection (WIfI) score is unknown. The aims of this study were to compare the reproducibility, completion times and ability to predict 30-day amputation of the WIfI, University of Texas Wound Classification System (UTWCS), Site, Ischemia, Neuropathy, Bacterial Infection and Depth (SINBAD) and Wagner classifications systems using photographs of diabetes-related foot ulcers.Methods:Three trained observers independently scored the diabetes-related foot ulcers of 45 participants on two separate occasions using photographs. The inter- and intra-observer reproducibility were calculated using Krippendorff’s α. The completion times were compared with Kruskal-Wallis and Dunn’s post-hoc tests. The ability of the scores to predict 30-day amputation rates were assessed using receiver operator characteristic curves and area under the curves.Results:There was excellent intra-observer agreement (α >0.900) and substantial agreement between observers (α=0.788) in WIfI scoring. There was moderate, substantial, or excellent agreement within the three observers (α>0.599 in all instances except one) and fair or moderate agreement between observers (α of UTWCS=0.306, α of SINBAD=0.516, α of Wagner=0.374) for the other three classification systems. The WIfI score took significantly longer (P<.001) to complete compared to the other three scores (medians and inter quartile ranges of the WIfI, UTWCS, SINBAD, and Wagner being 1.00 [0.88-1.00], 0.75 [0.50-0.75], 0.50 [0.50-0.50], and 0.25 [0.25-0.50] minutes). None of the classifications were predictive of 30-day amputation (P>.05 in all instances).Conclusion:The WIfI score can be completed with substantial agreement between trained observers but was not predictive of 30-day amputation.